A polypeptide targeting p85 and preparation method and application thereof

Abstract

The present application relates to a kind of polypeptide targeted to p85 beta, specifically, the polypeptide has the amino acid sequence shown in SEQ ID NO:1, the polypeptide has high binding affinity with p85 beta and p85 alpha, destroys the spatial co-localization of p85 beta and POLR1A and p85 alpha, p85 beta and p110 alpha, promotes the degradation of POLR1A and p110 alpha, to inhibit the tumorigenesis of HCC.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a peptide targeting p85, its preparation method, and its application. Background Technology

[0002] Liver cancer, often called the "silent killer," poses a serious health challenge worldwide. Early symptoms of liver cancer are often subtle, and most patients are diagnosed at an advanced stage, missing the optimal window for treatment.

[0003] The dangers of liver cancer should not be underestimated. First, it seriously threatens the patient's life and health. As the disease progresses, patients experience severe abdominal pain, bloating, nausea, and poor appetite, leading to a significant decline in their quality of life. For patients in the middle and late stages, even with treatment, the survival period is relatively limited, and the treatment process is often accompanied by a huge economic and psychological burden. Furthermore, the cost of liver cancer treatment is exorbitant, ranging from tens of thousands to hundreds of thousands of yuan, which is undoubtedly a heavy financial burden for many families.

[0004] Liver cancer is highly malignant, and its occurrence and development mechanisms are not fully understood. Treatment options are limited, and the prognosis is relatively poor. Although many advances have been made in the treatment of liver cancer in recent years, such as the rise of targeted therapy and immunotherapy, the effectiveness of these new therapies still varies from person to person, and not all patients can benefit from them.

[0005] Therefore, there is an urgent need in this field for a drug that inhibits the growth of liver cancer and liver cancer cells. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide that inhibits the growth of liver cancer cells, its preparation method, and its application.

[0007] In a first aspect, the present invention provides an improved peptide targeting p85β, said peptide being selected from the group consisting of:

[0008] (a) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0009] (b) A polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology (or identity) with the amino acid sequence shown in SEQ ID NO:1, and said polypeptide having the biological function shown in any one of SEQ ID NO:1-13; or

[0010] (c) A derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 to 3, more preferably 1 to 2) amino acid residues of the amino acid sequence shown in SEQ ID NO:1, and retaining the biological function shown in SEQ ID NO:1.

[0011] In another preferred embodiment, the polypeptide has one or more functions selected from the group consisting of:

[0012] (1) Inhibits tumor progression;

[0013] (2) Inhibit the activation activity of the PI3K / AKT signaling pathway;

[0014] (3) Downregulates the expression of POLR1A in cells;

[0015] (4) Inhibit p85β / POLR1A aggregates.

[0016] In another preferred embodiment, the polypeptide also targets p85α.

[0017] In a second aspect, the present invention provides a fusion polypeptide comprising the polypeptide described in the first aspect of the present invention.

[0018] In another preferred embodiment, the fusion polypeptide has one or more functions selected from the group consisting of:

[0019] (1) Inhibits tumor progression;

[0020] (2) Inhibit the activation activity of the PI3K / AKT signaling pathway;

[0021] (3) Downregulates the expression of POLR1A in cells;

[0022] (4) Inhibit p85β / POLR1A aggregates.

[0023] In another preferred embodiment, the N-terminus or C-terminus of the fusion polypeptide further contains a third fusion element, which is a transmembrane peptide (CPP) element.

[0024] In another preferred embodiment, the N-terminus or C-terminus of the fusion polypeptide further contains a fourth fusion element, which is a tumor-specific targeting element.

[0025] In another preferred embodiment, the targeting element is selected from the group consisting of antibodies, single-chain antibodies, polypeptides (including linear peptides and cyclic peptides), and immune molecules.

[0026] In a third aspect, the present invention provides a polynucleotide encoding a polypeptide as described in the first aspect of the present invention or a fusion polypeptide as described in the second aspect of the present invention.

[0027] In another preferred embodiment, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:2.

[0028] In another preferred embodiment, the polynucleotide has a homology (or identity) with the nucleotide sequence of SEQ ID NO:2 of ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.

[0029] In a fourth aspect, the present invention provides an expression vector containing the polynucleotides described in the third aspect of the present invention.

[0030] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0031] In another preferred embodiment, the vector is a retroviral vector.

[0032] In another preferred embodiment, the carrier is a linear or ring-shaped carrier.

[0033] In another preferred embodiment, the vector is a circular or linear RNA vector.

[0034] In another preferred embodiment, the vector further includes elements selected from the group consisting of: promoters, enhancers, transcriptional enhancer elements (WPREs), long terminal repeats (LTRs), selective markers, etc.

[0035] In another preferred embodiment, the vector comprises one or more promoters operably linked to polynucleotides as described in the third aspect of the invention.

[0036] In another preferred embodiment, the carrier comprises the sequence shown in formula (I):

[0037] A1-A2-A3-A4-A5(I)

[0038] In the formula, A1 is the Scar sequence;

[0039] A2 is a 3'UTR sequence;

[0040] A3 is a nucleotide sequence encoding a polypeptide as described in the first aspect of the present invention;

[0041] A4 is an IRES sequence;

[0042] A5 is a 5'UTR sequence;

[0043] The connection between A1 and A5 makes the carrier a ring-shaped carrier.

[0044] In a fifth aspect, the present invention provides a host cell containing an expression vector as described in the fourth aspect of the present invention, or having a genome containing polynucleotides as described in the third aspect of the present invention.

[0045] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0046] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0047] In a sixth aspect, the invention provides the use of the polypeptide as described in the first aspect or the fusion polypeptide as described in the second aspect for the preparation of a medicament for the prevention and / or treatment of tumors.

[0048] In another preferred embodiment, the tumor includes liver cancer.

[0049] In another preferred embodiment, the tumor is a tumor or cancer with activated PI3K / AKT signaling pathway.

[0050] In another preferred embodiment, the tumor is selected from the group consisting of: gastrointestinal cancer, cancer of the central or peripheral nervous system, cancer of the endocrine or neuroendocrine system or cancer of the hematopoietic system, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0051] In a seventh aspect, the present invention provides a pharmaceutical composition comprising:

[0052] (i) the polypeptide as described in the first aspect of the invention or the fusion polypeptide as described in the second aspect of the invention; and

[0053] (ii) Pharmaceutically acceptable carriers.

[0054] In another preferred embodiment, the pharmaceutical composition further includes a drug for treating tumors or inhibiting the activation activity of the PI3K / AKT signaling pathway.

[0055] In another preferred embodiment, the pharmaceutical composition is an injectable formulation.

[0056] In another preferred embodiment, (i) is encapsulated in a pharmaceutically acceptable carrier.

[0057] In another preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0058] In another preferred embodiment, the pharmaceutically acceptable excipient is selected from the group consisting of diluents, excipients, surfactants, lubricants, disintegrants, or combinations thereof.

[0059] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, oral formulations, and topical formulations.

[0060] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of oral, intravenous, and intraperitoneal injection.

[0061] In another preferred embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intratumoral, intramuscular, subcutaneous, or intraperitoneal injection.

[0062] In another preferred embodiment, the pharmaceutical composition is administered transdermally, such as by topical application or electrode delivery.

[0063] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for the prevention and / or treatment of cancer or tumors.

[0064] In an eighth aspect, the present invention provides a method for blocking the PI3K / AKT signaling pathway in tumor cells in vitro, comprising the steps of:

[0065] The tumor cells are treated with a polypeptide as described in the first aspect of the present invention or a fusion protein as described in the second aspect of the present invention, thereby blocking the PI3K / AKT signaling pathway in the tumor cells.

[0066] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0067] In another preferred embodiment, the tumor cells are selected from cells in tumor tissues from the group consisting of: gastrointestinal cancer, central or peripheral nervous system cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0068] A ninth aspect of the present invention provides a method for producing a polypeptide as described in the first aspect of the present invention, comprising the steps of:

[0069] (s1) Under conditions suitable for polypeptide production, host cells as described in the fifth aspect of the present invention are cultured to obtain a culture containing the polypeptide; and

[0070] (s2) Isolate or recover the polypeptide from the culture.

[0071] In another preferred embodiment, the polypeptide as described in the first aspect of the invention or the fusion polypeptide as described in the second aspect of the invention is synthesized by chemical synthesis.

[0072] In a tenth aspect, the present invention provides a method for treating and / or alleviating tumors, comprising the steps of:

[0073] The polypeptide, the fusion polypeptide, or the pharmaceutical composition as described in the first aspect of the present invention, the second aspect of the present invention, or the seventh aspect of the present invention are administered to the desired recipient.

[0074] In another preferred embodiment, the object is a mammal, such as a human, a mouse, or a monkey.

[0075] In another preferred embodiment, the subject suffers from a tumor or cancer.

[0076] In another preferred embodiment, the tumor is a tumor with activated PI3K / AKT signaling pathway.

[0077] In another preferred embodiment, the tumor is selected from the group consisting of: gastrointestinal cancer, cancer of the central or peripheral nervous system, cancer of the endocrine or neuroendocrine system or cancer of the hematopoietic system, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0078] In another preferred embodiment, the tumor is liver cancer.

[0079] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0080] Figure 1The study demonstrated that p85β promotes the development of HCC. a: Correlation analysis of protein levels between p110α and p85β was performed using the RPPA database; b: Correlation between PIK3R2 / p85β expression and overall survival in patients with different cancer types was analyzed using the TCGA database; c: High p85β expression was associated with poorer overall survival in HCC patients using tissue microarrays (HR: hazard ratio); df: Knockout of p85β inhibited HCC cell growth, and the knockout effect of control cells (shNC) and p85β knockout cells (shp85β) was examined. Proliferation rate (d), cck-8 proliferation rate (n=5) (e), and colony-forming ability (n=3) (f); gi: p85β knockout inhibits the growth of subcutaneous xenograft tumors, showing representative tumor images (g), growth curves (h), and final tumor weight (i) of subcutaneous xenograft tumors in specified cell lines (n=10); jl: p85β knockout inhibits the occurrence of spontaneous hepatocellular carcinoma, wild-type (Ctrl) and Pik3r2 / p85β conditional knockout (cKO) mice were injected with AAV8 TGB-Cre virus, followed by injection of myr-AKT1 and NRasV12 plasmids, showing representative liver tumor images and liver weight with tumor burden (Ctrl=19, cKO=12) (j); Overall survival of Ctrl and cKO mice was plotted (k). IHC staining of p85β and Ki67 in liver tumors (l).

[0081] Figure 2 This study demonstrates that p85β nuclear translocation is crucial for its oncogenic function in HCC. a: p85β knockout has no effect on AKT signaling and H3K27me3 in HCC cells; bd: Untranslocated p85β cannot rescue the growth defects caused by p85β knockout, schematic diagram of p85β nuclear translocation blocking mutation (b); wild-type p85β rescues the proliferation (c) and colony formation (d) of p85β knockout HCC cells, but untranslocated p85β cannot; ei: blocking p85β nuclear translocation inhibits the occurrence of spontaneous hepatocellular carcinoma. Phosphorylation sites and nuclear localization signal sequences regulating p85β nuclear translocation are identical between human p85β (hp85β) and mouse p85β (e); DNA sequences around mPik3r2Y458 in wild-type (Pik3r2+ / +) mice and homozygous mutant knock-in (Pib3r2YF / YF) mice (f); images of representative liver tumors and liver weights with tumor burden (Pi3kr2+ / - = 14, Pi3kr2YF / YF = 11) (g); IHC staining of p85β and Ki67 in liver tumors (h).

[0082] Figure 3This study demonstrates that phase separation of p85β in the cell nucleus promotes the development of HCC. a: p85β forms liquid-like droplets in the nucleus of HCC cells; bd: p85β undergoes LLPS; recombinant p85β-EGFP or p85β-mHerry fusion protein forms liquid droplets in vitro (b); p85β-EGFP forms droplets in the nucleus of Huh7 cells (c), and the droplets rapidly recover after photobleaching (FRAP assay) (d); ei: iSH2 and cSH2 domains are important for the phase separation of p85β; schematic diagram of p85β truncated proteins with different domains (e). The C-terminus of p85β, including nSH2, iSH2, and cSH2, exhibits phase separation ability (f, g); the coexistence of iSH2 and cSH2 exhibits phase separation (h, i); j-1: the loss of cSH2 in p85β disrupts the LLPS of p85β and cannot salvage the growth defects caused by p85β knockout; p85β-Δc cannot form droplets in the nucleus of HCC cells (j); overexpression of p85β-Δc cannot restore the cell proliferation (k) and colony formation (l) defects caused by p85β knockout.

[0083] Figure 4 The co-localization of p85β with POLR1A in the nucleolar fibrous center was shown. ac: p85β interacts with the Pol I subunit POLR1A; the interacting proteins of p85β in 293T cell nuclear lysates were identified by mass spectrometry (a), and the interaction between p85β and POLR1A was verified by immunoprecipitation of p85β (b) or POLR1A (c) with HCC cell nuclear lysates; dg: p85β co-localizes with POLR1A in the nucleolar fibrous center; co-immunofluorescence staining of p85β with nucleolar-labeled nucleolin (d), FC (fibrous center)-labeled POLR1A (e), DFC (dense fibrous component)-labeled FBL (f), or GC (granular component)-labeled PES1 (g); h: p85β and POLR1A co-localize in vitro; p85β-EGFP and POLR1A-y3 proteins were purified in vitro, and the phase separation of p85β and POLR1A was analyzed.

[0084] Figure 5This study demonstrates that p85β stabilizes POLR1A to enhance the stemness of HCC cells. ac: p85β stabilizes POLR1A; p85β knockdown reduces POLR1A protein levels (a) but does not regulate POLR1A mRNA levels (b); p85β knockdown increases POLR1A ubiquitination (c); dh: p85β knockdown inhibits rRNA synthesis, thereby reducing total protein synthesis; p85β knockdown reduces 45S pre-rRNA levels in HCC cells under various tumor microenvironments (d); p85β knockdown inhibits total RNA synthesis as measured by EdU (e, f); p85β knockdown inhibits total protein synthesis as measured by OPP (g, h); il: p85β knockdown inhibits HCC cell stemness; after p85β knockdown, the percentage of CD133-positive cells (i) and CD90-positive cells (j) decreases; after p85β knockdown, the diameter of organoids formed by HCC cells decreases (k, l).

[0085] Figure 6 The images show how peptides encoding the p110-α ABD domain block p85β nuclear translocation and inhibit HCC tumor growth. a, b: Peptides containing the p110α-ABD domain block p85β nuclear translocation; protein structure of the interface between the p110α-ABD domain and the p85β iSH2 domain (structural data from a protein database, ID: 7PG5) (a); overexpression of ABD-EGFP blocks the co-localization of nuclear p85β and POLR1A (b); c: Schematic diagram of the circRNA encoding the ABD peptide (circABD); d: circABD treatment reduces the protein levels of POLR1A and p110α; e: circABD treatment reduces HCC. Cell survival rate; fh: circABD treatment induced HCC cell apoptosis, and RNA sequencing analysis showed that circABD increased cell apoptosis (f, g), and circABD treatment significantly induced HCC cell apoptosis (h); ij: circABD treatment inhibited the growth of xenograft tumors in MHCC97H cells, and the flowchart of the circABD treatment xenograft tumor model (i); Representative tumor images and tumor weight were assessed after mouse sacrifice (i); IHC staining of Ki67, POLR1A, and PI3K / AKT signaling pathways was analyzed (j).

[0086] Figure 7 This shows that p85β promotes the development of HCC. a: IHC staining of Ki67 in control and p85β knockdown MHCC97H cell xenograft tumors; b: Schematic diagram of Pik3r2 conditional knockout mouse strain.

[0087] Figure 8This shows the generation of a mutant mouse strain that blocks p85β nuclear entry. Strategy for the Pik3r2 Y458F point mutation knockout mouse strain (a). The mRNA (b) and protein (c) levels of Pik3r2 / p85β were not affected by the Pik3r2 mutation. Y458F Change due to mutation.

[0088] Figure 9 The study showed that p85β stabilizes POLR1A. Wild-type p85β rescued POLR1A protein levels, but non-nuclear p85β could not restore them (a); USP7 knockout reduced POLR1A protein levels (b); MG132 inhibited ubiquitination and rescued POLR1A protein levels after USP7 knockdown (c); the correlation between p85β and POLR1A in patient tissue microarrays was shown (d).

[0089] Figure 10 This study demonstrates that p85β regulates the stemness of HCC cells via POLR1A. ac: Knockdown of POLR1A inhibits HCC cell proliferation; POLR1A knockout efficiency (a), CCK-8 proliferation rate (b), and colony formation (c) were measured; the proportion of CD133-positive (d) or CD90-positive (e) HCC cancer stem cells was also measured.

[0090] Figure 11 The study showed that peptides encoding the p110α-ABD domain inhibited HCC tumor growth. ad: Delivery of the p110α-ABD domain inhibited HCC tumor growth; overexpression of the p110α-ABD domain reduced POLR1A protein levels in HCC cells (a); AAV-ABD treatment inhibited xenograft tumor growth in MHCC97H cells (b); Tumor volume was measured before and after AAV-ABD treatment (c); AAV-ABD treatment reduced Ki67-positive cell and POLR1A expression in xenograft tumors (d); eh: circRNA encoding p110α-ABD inhibited tumor growth; in vitro synthesis of circular RNA containing the p110α-ABD coding region (e); circABD was validated by RT-PCR (f); circABD expressed FLAG-labeled ABD at levels comparable to the plasmid vector (g); circABD treatment inhibited xenograft tumor growth in MHCC97H cells (h). Detailed Implementation

[0091] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a kinase-independent, phase-separation-mediated carcinogenesis mechanism of p85β in hepatocellular carcinoma (HCC). Specifically, p85β forms biomolecular condensates through liquid-liquid phase separation (LLPS) and specifically localizes to the fibrous center of the nucleolar in HCC cells. Within the nucleolar, p85β interacts with and stabilizes the structure of POLR1A, thereby enhancing rRNA biosynthesis and driving HCC progression. Based on the above mechanism, the inventors have developed a polypeptide that inhibits hepatocellular carcinoma (HCC). This polypeptide targets the p85β / POLR1A condensate and inhibits the PI3K / AKT signaling pathway. Specifically, the polypeptide has a high binding affinity for p85β, disrupting the spatial co-localization of p85β and POLR1A and promoting POLR1A degradation. Simultaneously, the polypeptide promotes p110α degradation by binding to p85α and p85β, thereby inhibiting HCC tumorigenesis and exhibiting excellent anti-tumor effects. This invention is based on this principle.

[0092] the term

[0093] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0094] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0095] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0096] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0097] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0098] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0099] As used herein, the term “recombinant” refers to a nucleic acid, vector, polypeptide, or protein produced by DNA recombination (cloning) methods and distinguishable from natural or wild-type nucleic acids, vectors, polypeptides, or proteins.

[0100] As used herein, the term "host cell" refers to, for example, microbial, yeast, insect, and mammalian cells that can or have been used as rAAV vector recipients. This term includes the progeny of the transduced original cell. Therefore, as used herein, "host cell" generally refers to a cell that has been transduced with a foreign DNA sequence. It should be understood that due to natural, accidental, or artificial mutations, the progeny of a single parent cell may not necessarily be morphologically identical or complementary to the original parent in terms of genome or total DNA.

[0101] As used in this article, "vector" refers to a recombinant plasmid or virus containing polynucleotides delivered to host cells in vitro or in vivo. "Recombinant" means different from what is normally found in nature.

[0102] As used herein, the term "promoter" refers to a nucleic acid sequence or segment that has the function of controlling the transcription of one or more genes (or coding sequences), is located upstream of the transcription start site of the gene, and is structurally identified as having a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to directly or indirectly regulate the amount of transcription from a promoter.

[0103] As used in this article, the term "enhancer" is a cis-acting element that stimulates or represses transcription of adjacent genes. Enhancers that repress transcription are also called "silencers." Enhancers can exert their effects (e.g., they can be associated with coding sequences) at distances of up to thousands of base pairs (kb) downstream of coding sequences and transcribed regions in either direction.

[0104] "Identity" refers to the sequence matching between two polypeptides or two nucleic acids. "Identity" represents the percentage of identical residues in the polypeptide or nucleic acid sequence out of the total number of residues, and is calculated based on mutation type. Mutation types include insertions (extensions) at either end of a sequence, deletions (truncations) at either end of a sequence, substitutions of one or more amino acids / nucleotides, insertions within a sequence, and deletions within a sequence.

[0105] For example, in polypeptide sequences, if the mutation type is one or more of the following: substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence, the total residue count is calculated based on the larger of the compared molecules. If the mutation type also includes insertions (extensions) or deletions (truncations) at either end or both ends of the sequence, the number of amino acids inserted or deleted at either end or both ends (e.g., less than 20 at either end) is not included in the total residue count. When calculating the percentage of identity, the sequences being compared are aligned in a manner that produces the maximum match between sequences, and gaps in the alignment (if present) are resolved using a specific algorithm.

[0106] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0107] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0108] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0109] This invention includes not only complete polypeptides, but also fragments of immunologically active polypeptides or fusion proteins formed by polypeptides and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said polypeptides.

[0110] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the polypeptide of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify the polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0111] The variants of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the coding DNA of the polypeptide of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the polypeptide of the present invention.

[0112] The present invention also provides a polynucleotide molecule encoding the aforementioned polypeptide or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0113] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0114] The term "polynucleotide encoding a polypeptide" can include a polynucleotide that encodes the polypeptide, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0115] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0116] The full-length nucleotide sequence or fragments of the polypeptides of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0117] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0118] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0119] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0120] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0121] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0122] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0123] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0124] PI3K / AKT signaling pathway

[0125] The phosphoinositol-3-kinase (PI3K) / AKT signaling pathway is one of the most commonly dysregulated pathways in human cancers. Hepatocellular carcinoma (HCC), the main form of primary liver cancer, is a leading cause of cancer-related morbidity and mortality worldwide, and is also associated with PI3K / AKT pathway dysregulation.

[0126] PI3K complex

[0127] The PI3K complex consists of regulatory subunits (p85α and p85β) and catalytic subunits (p110α, p110β, p110δ, and p110γ). Upon stimulation by growth factors or insulin, the PI3K complex is recruited to the cell membrane, converting phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). As a second messenger, PIP3 binds to and activates multiple downstream effectors, such as AKT and PDK1, through its pleckstrin homology (PH) domain, thereby initiating a series of signaling events driving tumorigenesis.

[0128] Regulatory subunit p85

[0129] The regulatory subunit p85 typically stabilizes the structure of p110 and modulates its lipid kinase activity, thereby participating in processes such as cell growth, migration, and apoptosis. However, p85 can also perform a variety of biological functions independently of the catalytic activity of p110. For example, p85α directly binds to PTEN, enhancing its lipid phosphatase activity. Furthermore, p85α can activate CDC42, mediating cytoskeleton remodeling through a PI3K-independent mechanism. Both p85α and p85β promote the nuclear translocation of XBP1 (X-box binding protein 1), thereby regulating endoplasmic reticulum stress responses. In addition, p85α and p85β mediate the interaction between BRD7 (bromine-containing domain protein 7) and XBP1 to maintain cellular glucose homeostasis. The nuclear translocation of p85β promotes tumorigenesis by stabilizing the histone methyltransferases EZH1 / EZH2. More than 30% of p85 exists independently of p110 in mouse livers, suggesting that p85 independent of p110 may play an important role in liver physiological functions, including HCC progression.

[0130] The polypeptides of the present invention and their applications

[0131] In this invention, the terms "polypeptide of the present invention" and "p110α-ABD peptide" are used interchangeably and both refer to a polypeptide having the amino acid sequence shown in SEQ ID NO:1. The polypeptide of the present invention can achieve the following functions: (1) inhibiting tumor progression; (2) inhibiting the activation activity of the PI3K / AKT signaling pathway; (3) downregulating the expression of POLR1A in cells; and / or (4) inhibiting p85β / POLR1A condensates.

[0132] Based on common knowledge in the art, those skilled in the art will know that in non-critical regions of a polypeptide, such as its ends, altering a few amino acid residues will not substantially change its biological activity. For example, appropriately substituting certain amino acids will not affect the resulting sequence (see Watson et al., Molecular Biology of The Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224). Therefore, those skilled in the art can implement such substitutions and ensure that the resulting molecule retains the desired biological activity.

[0133] Therefore, based on the teachings of this invention, it is obvious to those skilled in the art that mutants of the polypeptides of this invention can be obtained by mutation, thereby retaining the desired function and activity. For example, it is known to those skilled in the art that adding or removing several amino acid residues at either end of the polypeptide, such as preferably 1-6, more preferably 1-3, and most preferably 1 amino acid residue, will not affect the function of the resulting mutant.

[0134] Therefore, this invention should include conserved mutants of the polypeptides of this invention. These conserved mutants can be generated by, for example, amino acid substitutions as shown in Table A below.

[0135] Table A

[0136]

[0137]

[0138] The present invention also provides polynucleotides encoding the polypeptides of the present invention. The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding this polypeptide, or may include polynucleotides that also include additional coding and / or non-coding sequences.

[0139] Preparation of peptides

[0140] The DNA sequences of the polypeptides or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single-chain polypeptide.

[0141] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0142] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0143] Currently, the DNA sequence encoding the polypeptide (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0144] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0145] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0146] Typically, host cells transformed with the present invention are cultured under conditions suitable for the expression of the polypeptides of the present invention. The polypeptides of the present invention are then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, and other conventional separation and purification methods well known to those skilled in the art.

[0147] The obtained monoclonal peptides can be identified using conventional methods. For example, the binding specificity of monoclonal peptides can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal peptides can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0148] The polypeptides of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0149] Pharmaceutical Composition

[0150] Based on the polypeptide of the present invention, the present invention further provides an antitumor pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0151] As used herein, the term "effective dose" or "therapeutic effective dose" refers to a dose that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0152] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0153] The pharmaceutical compositions of the present invention contain a safe and effective amount of the polypeptide of the present invention as the active ingredient and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration. The dosage form of the pharmaceutical compositions of the present invention can be a solid or solution form, preferably a solution form, such as an injection, oral formulation (tablets, capsules, oral liquid), transdermal formulation, or sustained-release formulation. For example, they can be prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0154] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Depending on the treatment requirements, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0155] The pharmaceutically acceptable carriers, effective amounts of active ingredients, and methods of administration described in this invention are well known to those skilled in the art.

[0156] Preferably, the polypeptides of the present invention can be used for tumor treatment selected from the group consisting of: gastrointestinal cancer, central or peripheral nervous system cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0157] Preferably, the polypeptides of the present invention can be used for the treatment of immune-related diseases selected from the group consisting of: rheumatoid arthritis, systemic lupus erythematosus, scleroderma, myositis, Sjögren's syndrome, immune thrombocytopenic purpura, or combinations thereof.

[0158] Preferably, the polypeptides of the present invention can be used for the treatment of neurodegenerative diseases selected from the group consisting of multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or combinations thereof.

[0159] The main advantages of this invention include:

[0160] 1. This application discloses a kinase-independent, phase-separation-mediated oncogenic mechanism of p85β in hepatocellular carcinoma (HCC). p85β forms biomolecule condensates via liquid-liquid phase separation (LLPS) and specifically localizes to the fibrous center of the nucleolus in HCC cells. Within the nucleolus, p85β interacts with and stabilizes the structure of POLR1A, thereby enhancing rRNA biosynthesis and driving HCC progression.

[0161] 2. The peptides of the present invention target p85β / POLR1A condensates and inhibit the PI3K / AKT signaling pathway, and have excellent anti-tumor effects.

[0162] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0163] 1. Method

[0164] 1.1 Circular RNA Synthesis

[0165] Template synthesis: The T7 promoter, upstream of the T4 td gene intron, 5' untranslated region, IRES, ABD coding sequence, 3' untranslated region, and downstream of the T4 td gene intron are linked in the direction from 5' to 3' end.

[0166] Transcription: Prepare the transcription system (T7 RNA transcriptase, 1X T7 RNA transcriptase buffer, ATP, CTP, GTP, UTP (20mM each), template (500ng)). Incubate at 37℃ for 16h using a PCR instrument.

[0167] Purification: Add DNase and RNase R to the system and incubate at 37°C for 1 hour. Purify the circular RNA using LiCl and anhydrous ethanol.

[0168] Detection: The purity of the product was determined by agarose gel electrophoresis and ultraviolet spectrophotometry.

[0169] 1.2 Preparation of AAV8

[0170] The target sequence was cloned into the GPAAV-CMV-WPRE expression plasmid, and the plasmid was then detoxified. Purity and concentration were determined using a UV spectrophotometer.

[0171] The expression plasmid, AAV8 serotype plasmid, and helper plasmid pHelper vector were transfected into AAVPro-293T cells at a ratio of 1:1:2 using HGtransgene reagent. The culture medium was changed after 6–8 hours of culture (all culture media were sterilized with fetal bovine serum).

[0172] After culturing for 72 hours, the cell suspension was repeatedly transferred between a dry ice foam box containing ethanol and a 37°C water bath, undergoing three freeze-thaw cycles. Centrifuged at 4000 rpm for 10 minutes. 2) The supernatant after centrifugation was transferred to a supernatant tube and centrifuged at 10 WG for 2 hours. 3) Benzonase (total 50 U / mL) and MgCl2 (total 0.002 M) were added and centrifuged at 37°C for 20 minutes. 4) The supernatant was filtered through a 0.22 μm filter and transferred to a horizontal supernatant tube. 5) A gradient of 60%, 40%, 25%, and 15% iodixanol was prepared and balanced. 6) Centrifuged at 27.5 WG and 4°C for 4 hours. 7) Transfer to a 50KD ultrafiltration tube, centrifuge at 4500 rpm to the desired viral load, and discard the bottom portion of waste liquid. 8) Aliquot and store the virus. 9) After aliquoting, store the virus at -80℃.

[0173] 1.3p85β and POLR1A localization detection

[0174] Immunohistofluorescence or cellular immunofluorescence (fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton-X100) was used to bind anti-p85β (abcam, Cat#ab180967) and POLR1A (stana cruz, sc-48385), followed by color development with fluorescent secondary antibody. Detection was then performed using fluorescence confocal microscopy.

[0175] 2. Results

[0176] 2.1 PIK3R2 / p85β promotes HCC progression

[0177] Given that p85, independent of p110, has been found in normal liver, we set out to investigate the function and potential mechanisms of p85β, independent of p110, in hepatocellular carcinoma (HCC). Consistent with current understanding, p85 and p110α protein levels are highly correlated in most cancers, based on TCGA protein array (RPPA) data. However, the correlation between p85 and p110α protein levels is weaker in LIHC (hepatocellular carcinoma), as well as LUSC (lung squamous cell carcinoma), READ (rectal adenocarcinoma), and TGCT (testicular germ cell tumor) (Fig. 1a). In these four tumors, only in LIHC were high levels of PIK3R2 / p85β associated with poorer overall survival (Fig. 1b), suggesting that p85β, independent of p110, may play an important role in tumorigenesis in LIHC.

[0178] This further confirms the oncogenic function of p85β in HCC development. Firstly, knockdown of p85β significantly inhibited the proliferation and colony-forming ability of HCC cells. Figure 1 d-1f). Secondly, knockdown of p85β significantly reduced the growth rate and final weight of xenograft subcutaneous tumors generated by HCC cells (d-1f). Figure 1 Finally, we established a Pik3r2 conditional knockout mouse strain and validated the function of p85β using a spontaneous HCC model. Figure 7 b). Wild-type (Ctrl) and Pik3r2 conditional knockout (cKO) mice were injected with AAV8-TGB-Cre virus, followed by tail vein injection of activated NRasV12 (N-Ras G12V) and AKT1 (myr-AKT1) to induce HCC ( Figure 1 Comparative analysis showed that Pik3r2 cKO mice exhibited lower liver tumor burden and liver weight (j). Figure 1 j), and their survival time was prolonged compared to the control group mice (j). Figure 1 Immunohistochemical results confirmed the knockout efficiency of p85β and the reduced proliferative capacity in cKO mice. Figure 1 These data suggest that p85β plays an oncogene role in HCC progression.

[0179] 2.2 Nuclear localization: p85β dominates its oncogenic function

[0180] Although p85β is known to regulate the classical AKT signaling pathway in the cytoplasm or regulate histone methyltransferases EZH1 / 2 in the nucleus to promote tumorigenesis, its specific role in hepatocellular carcinoma (HCC) remains unclear. In HCC cells, knockdown of p85β did not affect the phosphorylation levels of AKT, mTOR, GSK3β, and ERK, nor did it alter the protein levels of EZH1, EZH2, and H3K27me3. Figure 2 a). To further investigate whether p85β in the cytoplasm or nucleus plays a carcinogenic role in HCC, the rescue capabilities of various p85β mutants for growth defects induced by p85β knockdown were evaluated. Figure 2 b). Notably, only overexpression of wild-type p85β could restore the proliferation and colony-forming abilities of p85β-knockout cells, while p85β mutants that cannot enter the nucleus (p85βY464F or p85βNLSmut) could not restore these abilities. Figure 2 (c-2d). This highlights the crucial role of intranuclear p85β in HCC tumorigenesis.

[0181] Nuclear translocation of human p85β (hp85β) depends on phosphorylation at Y464 and the nuclear localization signal (NLS) sequence near Y464. Mouse p85β (mp85β) has the same tyrosine residue and NLS sequence at Y458. Figure 2 e). To further verify the oncogenic function of nuclear p85β, we constructed a sample with the allelic point mutation Pik3r2. Y458F Transgenic mouse strains ( Figure 2 f and Figure 8 a) Following injection of NRasV12 and AKT1, a homozygous mutant knock-in of Pi3kr2 YF / YF Mice and wild-type (Pi3kr2) + / + Compared to mice, they exhibited lower liver tumor burden and liver weight. Figure 2 g). Importantly, Pi3kr2 YF / YF The levels of Pik3r2 / p85β mRNA and protein in mice are related to Pi3kr2. + / + Mouse-like ( Figure 8 bc), but nuclear translocation of p85β is reduced in mouse liver ( Figure 2 These data suggest that p85β promotes HCC tumorigenesis through its intranuclear form, independent of p110.

[0182] 2.3p85β forms droplet-like structures through liquid-liquid phase separation.

[0183] p85β tends to form liquid-like droplets within the nucleus of HCC cells. Figure 3a). To validate this observation, p85β expression vectors fused with EGFP or mCherry were constructed, and these recombinant proteins were purified for in vitro analysis. Both p85β-EGFP and p85β-mCherry formed liquid droplets in vitro. Figure 3 b). Similarly, p85β-EGFP droplets were also observed in the nucleus of HCC cells. Figure 3 c) and exhibited rapid recovery after photobleaching (FRAP experiment) Figure 3 (c and 3d). These findings indicate that p85β undergoes liquid-liquid phase separation both in vitro and in vivo.

[0184] To determine the domains driving liquid-liquid phase separation (LLPS) of p85β, a series of EGFP-fused p85β truncated mutants were constructed, and their phase separation capabilities were evaluated. Figure 3 e). First, the C-terminus of p85β (including the nSH2, iSH2, and cSH2 domains) exhibits phase separation capability, while the N-terminus does not. Figure 3 (f and 3g). Surprisingly, individual SH2 domains (nSH2, iSH2, or cSH2) cannot independently form liquid droplets. Figure 3 h). However, when testing combinations of two SH2 domains, droplets could only form when both iSH2 and cSH2 were present. Figure 3 Similarly, icSH2 fusion with EGFP can also form liquid droplets (h). Figure 3 i). It is worth noting that p85β can form liquid droplets at concentrations as low as 50 nM, while high concentrations of NaCl will disrupt p85β condensates. Figure 3 g and 3i).

[0185] To further investigate the functional relevance of LLPS, a p85β mutant lacking the cSH2 domain (p85β-Δc) was constructed. Figure 3 e). Unlike wild-type p85β, p85β-Δc is uniformly distributed within the nucleus of HCC cells and does not form visible droplets. Figure 3 Furthermore, overexpression of p85β-Δc failed to restore the proliferation and colony formation defects induced by p85β knockdown (j). Figure 3 These results indicate that phase separation of p85β within the nucleus is crucial for its oncogenic function in HCC tumorigenesis.

[0186] 2.4 In HCC cells, p85β and POLR1A co-localize within the nucleolus.

[0187] To investigate the composition of p85β condensates in the nucleus, the nuclei of 293T cells were isolated to identify potential interaction partners of p85β. Mass spectrometry analysis revealed that POLR1A, the largest subunit of RNA polymerase I (Pol I) in the nucleolus, is an important candidate molecule. Figure 4 a). Through immunoprecipitation experiments, we confirmed the interaction between POLR1A and p85β in hepatocellular carcinoma cells (a). Figure 4 (b and 4c).

[0188] The nucleolus, the largest non-membranous organelle in the eukaryotic cell nucleus, is primarily responsible for the large-scale synthesis of ribosomal RNA (rRNA) and the assembly of ribosomal subunits. It comprises three characteristic subregions—the fibrous center (FC), the dense fibrous fraction (DFC), and the granular fraction (GC)—which assemble into a complex and ordered structure through phase separation. As expected, co-localization analysis using the nucleolar marker protein nucleolin revealed that p85β condensates are located within the nucleolus. Figure 4 d). The p85β condensate is precisely located at the center of the nucleolar filament (completely colocalized with the FC marker POLR1A) and surrounded by the DFC marker FBL and the GC marker PES1. Figure 4 e-4g). In vitro experiments also confirmed that the recombinantly expressed p85β-EGFP and POLR1A-AF555 proteins were almost completely co-localized. Figure 4 Notably, POLR1A failed to spontaneously form liquid-liquid phase separations (LLPS) in vitro, suggesting that phase separation within the nucleolar FC may require the involvement of other Pol I subunits or interacting partners. Upon increasing p85β protein concentration, recombinant POLR1A gradually formed droplets, indicating that high levels of p85β may promote the aggregation of POLR1A into dense FC-like structures. Figure 4 Furthermore, co-localization of POLR1A and p85β was also detected in other cancer cell lines. Figure 9 (a) suggests that p85β plays a universal role in nucleolar function across different types of cancer.

[0189] In summary, p85β plays a key role in the regulation of nucleolar organization by interacting with POLR1A and participating in the phase separation process.

[0190] 2.5p85β stabilizes the POLR1A structure via USP7.

[0191] p85β prevents its bound proteins from undergoing ubiquitination-mediated degradation in the nucleus by recruiting the deubiquitinating enzyme USP7. p85β may regulate POLR1A stability through this mechanism. As expected, knockdown of p85β specifically reduced POLR1A protein levels, but had no effect on POLR1C, another subunit of RNA Pol I. Figure 5 a). Knocking down p85β had no effect on POLR1A mRNA levels. Figure 5 b), but significantly increased the ubiquitination level of POLR1A protein ( Figure 5 c). Overexpression of wild-type p85β significantly restored POLR1A protein levels, while the p85β mutant, which cannot enter the nucleus, could not. Figure 9 a) This indicates that p85β regulates the stability of POLR1A within the nucleus. p85β stabilizes POLR1A by recruiting USP7, as knockdown of USP7 significantly reduces the protein level of POLR1A. Figure 9 b). Treatment with the proteasome inhibitor MG132 significantly restored POLR1A protein levels reduced by USP7 knockdown. Figure 9 c). Furthermore, in HCC tissues, positive staining for POLR1A was positively correlated with high expression of p85β. Figure 9 d).

[0192] 2.6p85β enhances HCC cell stemness via POLR1A

[0193] Based on the stabilizing effect of p85β on POLR1A, it is hypothesized that p85β may regulate the activity of RNA polymerase I (Pol I). Experimental results showed that under various tumor microenvironments, including acidic, hypoxic, amino acid-restricted, or glucose-restricted conditions, knockdown of p85β significantly reduced the transcription level of precursor rRNA (45S rRNA). Figure 5 d). Furthermore, p85β knockdown significantly inhibited rRNA synthesis ( Figure 5 e and 5f), which may lead to a decrease in global protein synthesis levels by impairing ribosome assembly (e and 5f). Figure 5 g and 5h).

[0194] To further verify whether p85β promotes liver cancer development through POLR1A, it was found that POLR1A knockdown, similar to p85β knockdown, significantly inhibited the proliferation and colony formation of liver cancer cells. Figure 10 a-10c). Notably, high expression of POLR1A has been reported as an important marker of stem cell hierarchy in colorectal cancer. By detecting hepatocellular carcinoma stem cell markers, we found that p85β knockdown significantly reduced the proportion of CD133+ or CD90+ hepatocellular carcinoma cells (a-10c). Figure 5i, 5j, S5d and S5e), and significantly reduced tumor spheroid size in 3D culture systems. Figure 5 These data indicate that p85β enhances hepatocellular carcinoma stemness and promotes tumorigenesis by regulating POLR1A.

[0195] 2.7p110α-ABD peptide inhibits HCC progression by targeting the p85β droplet-like structure.

[0196] Given the crucial role of p85β nuclear translocation in the progression of hepatocellular carcinoma (HCC), blocking this process has become a potential therapeutic strategy. Both p85β and p85α bind tightly to the ABD domain of p110α, which is primarily located in the cytoplasm. Figure 6 a). Based on this, we designed a peptide containing the p110α-ABD domain (FLAG-ABD-GFP) and overexpressed it in cells. Experimental results showed that the co-localization of p85β and POLR1A was significantly disrupted in cells expressing FLAG-ABD-GFP, while no similar phenomenon was observed in cells lacking GFP signaling. Figure 6 b). FLAG-ABD overexpression led to a significant downregulation of POLR1A expression. Figure 11 a) This replicated the effect of p85β knockdown or blockade of its nuclear translocation. Delivery of FLAG-ABD via adeno-associated virus (AAV) significantly inhibited the growth of MHCC97H-derived xenograft tumors. Figure 11 b and 11c), and reduced the proportion of Ki67+ cells and POLR1A expression in tumor tissue (b and 11c). Figure 11 d). These findings suggest that the p110α-ABD peptide can inhibit HCC progression by targeting the p85β / POLR1A condensate.

[0197] 2.8 Delivery of circRNA encoding p110α-ABD peptide in HCC cells can simultaneously inhibit the cellular p85β / POLR1A condensate and PI3K / AKT pathway.

[0198] To facilitate clinical application, the inventors explored various ABD peptide delivery strategies and ultimately selected circular RNA (circABD), which exhibits excellent stability and expression efficiency, as the expression vector for the FLAG-ABD peptide. Figure 6 c, 11e, and 11f). In vitro synthesized circABD can efficiently express the FLAG-ABD peptide in cells, achieving expression levels comparable to those of the plasmid vector. Figure 11 g), and significantly downregulated POLR1A protein expression ( Figure 6 d). Notably, circABD treatment also significantly reduced p110α protein levels and AKT phosphorylation ( Figure 6d). This effect may stem from circABD's ability to bind large amounts of p85 protein, reducing the p85 / p110α ratio and thus disrupting p110α's stability. These results indicate that circABD has a dual inhibitory function—targeting both the p85β / POLR1A condensate and blocking the PI3K / AKT signaling pathway.

[0199] Further evaluation of the therapeutic potential of circABD revealed that circABD treatment significantly inhibited liver cancer cell viability and induced apoptosis. Figure 6 e and 6f); RNA sequencing analysis confirmed that it works by activating the apoptosis pathway. Figure 6 g and 6h). In in vivo experiments, circABD inhibited the growth of MHCC97H-derived xenograft tumors (g and 6h). Figure 6 i and 11h), and reduced the expression of POLR1A, p110α and AKT phosphorylation levels in tumor tissue (i and 11h). Figure 6 (j). These data provide experimental evidence for circABD as a novel molecular intervention strategy for the treatment of liver cancer.

[0200] 3. Discussion

[0201] This application reveals the function and molecular mechanism of p85β in the development of hepatocellular carcinoma (HCC). Although the oncogenic role of p85β is widely recognized, the exact mechanism of its pro-tumorigenic function remains controversial. Previous studies have shown that cytoplasmic p85β promotes tumorigenesis in lung, colon, and breast cancer by regulating PI3K activity. However, in many normal tissues, the abundance of regulatory subunits p85α and p85β exceeds that of catalytic subunits p110α and p110β, resulting in the presence of p85β independent of p110. Notably, p85β is further overexpressed in multiple cancer types and associated with poor prognosis, suggesting that more p85β independent of p110 may exist in human cancers, and its function may be independent of PI3K regulation. In PIK3CA helical domain mutant tumors and clear cell renal cell carcinoma (ccRCC), nuclear p85β drives tumorigenesis by regulating histone methylation in a PI3K-independent manner. These findings demonstrate that p85β, independent of p110, plays an oncogenic role in the nucleus of HCC cells. These findings underscore the necessity for further investigation into the molecular mechanisms and therapeutic targeting of p85β, independent of p110, in cancer biology.

[0202] Although p85 proteins have long been considered regulatory subunits of PI3K, new evidence suggests they also function as adaptor proteins in a variety of PI3K-independent complexes. For example, the nSH2 mutant of p85α enhances HER3 (ERBB3) interaction, stabilizing HER3 to activate EGFR, HER2, and c-MET signaling pathways, thereby promoting tumorigenesis. Similarly, p85β stabilizes receptor tyrosine kinase AXL, inducing oncogenic signaling in ovarian cancer. In addition to receptor tyrosine kinase regulation, p85β depletion reduces BRD4 protein levels, impairing DNA damage repair. Furthermore, p85β and p85α can stabilize BRD7 protein to maintain glucose homeostasis, while nuclear p85β can stabilize EZH1 and EZH2 to promote cancer progression. Given the well-defined role of p85 proteins in stabilizing the p110 subunit, it is reasonable to infer that p85 proteins act as molecular adapters to regulate the stability of their interacting partners. In this application, p85β is presented as a novel aptamer protein, namely, stabilizing the RNA polymerase I subunit POLR1A and regulating rRNA synthesis during HCC progression. These findings highlight the importance of exploring p85-mediated protein degradation systems to further understand the mechanisms of p85 action independent of p110.

[0203] A novel mechanism by which the PI3K regulatory subunit p85β regulates nucleolar function. Strong evidence supports the role of p85β in the nucleolus. First, nuclear p85β is localized in the fibrous center of the nucleolus in HCC cells. Second, p85β interacts with and stabilizes the structure of POLR1A, a key component of the nucleolar fibrous center. Third, knockdown of p85β significantly impairs rRNA and global protein synthesis. Considering the strong binding affinity between p85 and p110, and the widespread presence of nuclear-localized p85 proteins, it can be inferred that p110 may also be present in the nucleus, and may even regulate biological processes within the nucleolus. In fact, Kumar et al. reported that p110β is located in the nucleus of MEF cells, while Mazloumi Gavgani et al. demonstrated the nucleolar localization of p110β and its product PIP3 in endometrial cancer cells. Therefore, AKT can be activated within the nucleus. Future research warrants investigation into whether PI3Kβ / AKT has a function in promoting cancer progression within the nucleolus.

[0204] This application reveals the crucial role of p85β phase separation in HCC progression. Although the dotted structure of p85 protein has been observed in various contexts, such as p85β in focal adhesion of 3T3 cells, p85α in IRS1-driven droplets during insulin signaling, and p85α / p110α in endosomes to activate AKT, no studies have yet demonstrated that p85 protein drives the formation of membraneless organelles via LLPS. In this application, it is demonstrated that p85β undergoes LLPS with POLR1A through intramolecular and intermolecular interactions. Although p85β is predicted to lack classic intrinsically disordered regions (IDRs) by IDR analysis software, the iSH2 and cSH2 domains are essential for p85β's LLPS. High concentrations of NaCl can disrupt p85β's LLPS, suggesting that electrostatic interactions may play a key role in p85β's LLPS. The conservation of the iSH2 and cSH2 domains in p85α and p85β suggests that p85α may also possess LLPS capability. Furthermore, given the pro-tumor effects of p85βLLPS, targeting p85β aggregates represents a promising therapeutic strategy for HCC treatment.

[0205] Although peptides are considered a strategy for disrupting LLPS structures, feasible and economical delivery of peptides into cells remains challenging. Messenger RNA (mRNA)-based vaccines have shown promising applications in infectious diseases and cancer because they are safer, do not infect or integrate, and can be synthesized rapidly and cost-effectively through in vitro transcription. Recent advances in RNA engineering further demonstrate that circular RNAs (circRNAs) possess greater stability and lower immunogenicity compared to linear mRNAs, thus expanding their potential in cancer therapy. This application found that a peptide containing the p110α-ABD domain has a high binding affinity for p85β. This peptide disrupts the spatial co-localization of p85β and POLR1A, promoting POLR1A degradation and thereby inhibiting HCC tumorigenesis. Finally, an optimized circRNA was developed to express the p110α-ABD peptide. Interestingly, the engineered circABD not only disrupts the p85β / POLR1A condensate but also inhibits the PI3K / AKT signaling pathway by promoting p110α degradation. This dual-function therapeutic strategy combines phase separation biology and oncogenic signaling pathways, significantly enhancing the therapeutic effect of HCC and positioning circABD as a paradigm-shifting approach in RNA-based cancer therapy.

[0206] sequence list

[0207]

[0208] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A polypeptide targeting p85β, characterized in that, The polypeptide is selected from the following group: (a) A polypeptide having the amino acid sequence shown in SEQ ID NO:1; (b) A polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology (or identity) with the amino acid sequence shown in SEQ ID NO:1, and said polypeptide having the biological function shown in any one of SEQ ID NO:1-13; or (c) A derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 to 3, more preferably 1 to 2) amino acid residues of the amino acid sequence shown in SEQ ID NO:1, and retaining the biological function shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide has one or more functions selected from the group consisting of: (1) Inhibits tumor progression; (2) Inhibit the activation activity of the PI3K / AKT signaling pathway; (3) Downregulates the expression of POLR1A in cells; (4) Inhibit p85β / POLR1A aggregates.

3. A fusion polypeptide, characterized in that, The fusion polypeptide comprises the polypeptide as described in claim 1.

4. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide as described in claim 1 or the fusion polypeptide as described in claim 3.

5. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 5, or its genome is integrated with the polynucleotide as described in claim 4.

7. The use of the polypeptide as claimed in claim 1, or the fusion polypeptide as claimed in claim 3, characterized in that, Used to prepare drugs for the prevention and / or treatment of tumors.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the polypeptide of claim 1 or the fusion polypeptide of claim 3; and (ii) Pharmaceutically acceptable carriers.

9. A method for blocking the PI3K / AKT signaling pathway in tumor cells in vitro, characterized in that, Including the following steps: The tumor cells are treated with the polypeptide of claim 1 or the fusion protein of claim 3 to block the PI3K / AKT signaling pathway in the tumor cells.

10. A method for producing the polypeptide as described in claim 1, characterized in that, Including the following steps: (s1) Under conditions suitable for polypeptide production, the host cell as described in claim 6 is cultured to obtain a culture containing the polypeptide; and (s2) Isolate or recover the polypeptide from the culture.

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