Compositions and methods for treating and preventing cancer

Abstract

Provided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6-phospate (G6PC) (e.g., by administering G6PC polypeptides) for treating cancer.

Description

COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING CANCER STATEMENT OF RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Patent Application No. 63 / 627,575, filed January 31, 2024, the entire contents of which are incorporated herein by reference for all purposes.STATEMENT OF GOVERNMENT SUPPORTThis invention was made with government support under grants no. DK 120309 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTINGThe text of the computer readable sequence listing filed herewith, titled “JHU-42665- 601_SQL.xml”, created January 22, 2025, having a file size of 65,589 bytes, is hereby incorporated by reference in its entirety.FIELDProvided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6- phospate (G6PC) (e.g., by administering G6PC nucleic acids or polypeptides) for treating cancer.BACKGROUNDCancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal bleeding, prolonged cough, unexplained weight loss, and a change in bowel movements. Over 100 types of cancers affect humans.Tobacco use is the cause of about 22% of cancer deaths. Another 10% are due to obesity, poor diet, lack of physical activity or excessive alcohol consumption. Other factors include certain infections, exposure to ionizing radiation, and environmental pollutants. In the developing world, 15% of cancers are due to infections such as Helicobacter pylori, hepatitis B, hepatitis C, human papillomavirus infection, Epstein-Barr virus and human immunodeficiency virus (HIV). These factors act, at least partly, by changing the genes of a cell. Typically, many genetic changes are required before cancer develops. Approximately 5- 10% of cancers are due to inherited genetic defects. Cancer can be detected by certain signs and symptoms or screening tests. It is then typically further investigated by medical imaging and confirmed by biopsy.The risk of developing certain cancers can be reduced by not smoking, maintaining a healthy weight, limiting alcohol intake, eating plenty of vegetables, fruits, and whole grains, vaccination against certain infectious diseases, limiting consumption of processed meat and red meat, and limiting exposure to direct sunlight. Early detection through screening is useful for cervical and colorectal cancer. The benefits of screening for breast cancer are controversial. Cancer is often treated with some combination of surgery, radiation therapy, surgery, chemotherapy and targeted therapy. Pain and symptom management are an important part of care. Palliative care is particularly important in people with advanced disease. The chance of survival depends on the type of cancer and extent of disease at the start of treatment. In children under 15 at diagnosis, the five-year survival rate in the developed world is on average 80%. For cancer in the United States, the average five-year survival rate is 66% for all ages.In 2015, about 90.5 million people worldwide had cancer. In 2019, annual cancer cases grew by 23.6 million people, and there were 10 million deaths worldwide, representing over the previous decade increases of 26% and 21%, respectively.The most common types of cancer in males are lung cancer, prostate cancer, colorectal cancer, and liver cancer. In females, the most common types are breast cancer, colorectal cancer, lung cancer, and cervical cancer. If skin cancer other than melanoma were included in total new cancer cases each year, it would account for around 40% of cases. In children, acute lymphoblastic leukemia and brain tumors are most common, except in Africa, where non-Hodgkin lymphoma occurs more often. In 2012, about 165,000 children under 15 years of age were diagnosed with cancer. The risk of cancer increases significantly with age, and many cancers occur more commonly in developed countries. Rates are increasing as more people live to an old age and as lifestyle changes occur in the developing world. The global total economic costs of cancer were estimated at US$1.16 trillion (equivalent to $1.56 trillion in 2022) per year as of 2010.New treatments for cancer are needed.SUMMARYProvided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6- phospate (G6PC) (e.g., by administering G6PC polypeptides) for treating cancer.Tumor cells exhibit glucose avidity for the great bioenergetic and biosynthetic demand. AMPK targeting-peptides can inhibit the growth of tumor cells through blockingAMPKal / 2 phosphorylation at S496 / 491, which can be directly phosphorylated by activated AKT, PKA, or AMPK itself, leading to the ubiquitination and degradation of aPKCi / 1, along with paradoxically increased CREB phosphorylation, subsequently the induction of glucose- 6-phosphatase (G6pc), the opposite of inhibited G6pc expression by activated AMPK in normal cells. Elevated G6PC removes the phosphate group from glucose 6-phosphate to limit its use by tumor cells. Overexpression of G6pc gene or treatment with G6PC nanoparticles inhibits the growth of tumor cells or hepatoma xenografts. Of particular interest, primary human hepatocytes or normal mammary epithelial cells can tolerate the treatment of G6PC nanoparticles or overexpression of G6pc gene. Data described herein support that G6PC may be a promising therapeutic agent for treating tumors.For example, in some embodiments, provided herein is a method of treating cancer in a subject, comprising: administering a composition (e.g., pharmaceutical composition) comprising glucose-6-phosphatase to the subject. In some embodiments, the glucose-6- phosphatase is glucose-6-phosphatase catalytic subunit 1, 2, or 3. The present disclosure is not limited to administering particular forms of G6PC. For example, in some embodiments, a G6PC polypeptide or a fragment thereof is administered. In some embodiments, a nucleic acid (e.g., DNA or RNA) encoding a G6PC is administered.The present disclosure is not limited to particular cancers. Examples include but are not limited to, breast cancer, liver cancer, melanoma, pancreatic cancer, lung cancer, brain cancer, or lymphoma.The present disclosure is not limited to a particular administration method. For examples, in some embodiments, the composition is administered locally to the site of the cancer. In some embodiments, the composition is administered systematically.In some embodiments, the glucose-6-phosphatase inhibits the growth of or kills tumor cells but not normal cells.Further embodiments provide the use of a composition comprising glucose-6- phosphatase to treat cancer in a subject.Additional embodiments provide a composition comprising glucose-6-phosphatase for use in treating cancer in a subject.Additional embodiments are described herein.DESCRIPTION OF THE FIGURESFIG. 1 shows induction of G6pc by Pa496h and Pa2-491 peptides decreases glucose utilization in tumor cells, a-d, HepG2 cells were treated with 300 pM TAT-control, Pa496hor Pa2-491 for 16 h, RNA-seq analysis was conducted. PC A was performed on differentially expressed genes (a). Heatmap shows the differential expression of transcriptomes (b), differential expression of genes and the fold change and statistical significance in cells treated with Pa496h verses TAT-control (c) or Pa2-491 verses TAT-control (d) (n=4 / group). e, f, HepG2 cells were treated with 300 pM TAT-control, Pa496h or Pa2-491 for 16 h. The relative mRNA levels of G6pc were determined (e) (n=3-4) and indicated protein levels were determined in immunoblots (f). g, MCF7 cells were treated with 300 uM of TAT or Pa2-491 for 16 h. h, i, HepG2 cells were treated with 300 pM of TAT and Pa496h for 16 h, oxygen consumption rate (OCR) (h) and extracellular acidification rate (ECAR) (i) were determined using Seahorse XF96 Extracellular Flux Analyzer (n=5 / group). *, p<0.05, one-way ANOVA. j-o, HepG2 cells (j), MCF-7 cells (k), HCC-DTCs (1), breast cancer DTCs (m), human primary hepatocytes (n), and human primary mammary epithelial cells (o) were treated with 200 pM of TAT or Pa496h for 16 h, then medium was changed to glucose-free DMEM containing 200 pM of fluorescence labelled 2-NBDG for 20 min, followed by washing with no color DMEM, and the fluorescence intensities were determined (n, counted cell number). *, p<0.05.FIG. 2 shows that G6PC reduces glucose utilization and inhibits the growth of tumor cells, a, Adenoviral expression vectors of GFP or G6pc were added to HepG2 cells for 48 h. b, c, Adenoviral expression vectors of GFP and G6pc gene were added to HepG2 cells (b) and MCF-7 cells (c), and cellular 2-NDBG levels were determined, d, e, Adenoviral expression vectors of GFP and G6pc gene were added to HepG2 cells (lxl0A10 vp) (d) and MCF-7 cells (lxlOA9 vp) (e) for 16 h (n=6). f, g, Adenoviral expression vectors were added to HCC-DTCs (2xlOA9 vp) (f) and human breast cancer DTCs (3xlOA9 vp) (g) for indicated time (n=6). h-j, Adenoviral expression vectors were added to human primary hepatocytes (2xlOA9 vp) (h) and human primary mammary epithelial cells (3xlOA9 vp) (i, j) for indicated time (n=6). k-p, Adenoviral expression vectors of GFP or G6pc were added to JIMT1 cells (3xlOA9 vp) (k), small cell lung cancer H69PR cells (lxlOA9 vp) (1), B-lymphoma Ramos cells (3xl0A9 vp) (m), melanoma HT144 (3xlOA9 vp) (n), and pancreatic adenocarcinoma BxPC3 cells (3xl0A9 vp) (o) for 40 h, and primary glioblastoma cells (5xlOA9 vp) for 16 h (p) (n=5-6 / group).FIG. 3 shows that treatment with G6PC nanoparticles inhibits the growth of tumor cells, a-d HepG2 cells (a, b) and MCF7 cells (c, d) were treated with 1 pM or 1.5 pM of nanoparticles vehicle and G6PC nanoparticles for 6 h respectively, OCR was determined (n=5-6). e-1, HepG2 cells (e), MCF7 cells (f), B-lymphoma Ramos cells (g), HEK293 cells(h), HCC-DTCs (i), breast cancer DTCs (j), human primary hepatocytes (k), and human primary mammary epithelial cells (1) were treated with 1 M M of nanoparticles vehicle and G6PC nanoparticles for 24 h (n=6). m-o, HepG2 cells (3-millions cells and VitroGel) together with 0.45 nM of NP-Vehicle or NP-G6PC were subcutaneously implanted into the flanks of male NSG mice, 0.45 nM of NP-G6PC or NP-control were injected directly into the tumor cells’ implanted sites at day 3, day 6, and day 9, then 0.9 nM of NP-G6PC or NP- control were injected directly into the tumor at day 12, 25, 18, 21, and 25 after implantation. Tumor xenografts were collected 4 weeks after the implantation. Tumor volume (m), size (n), and weight (o) were determined (n=5). p, q, HepG2 cell (p) and MCF7 cells (q) were treated with 300 pM of TAT control or Pa496h peptides for 16 h, followed by treatment with 20 pM of MG132 for 4 h. *, p<0.05.FIG. 4 shows G6PC1, 2, and 3 amino acid and nucleotide sequences.DEFINITIONSTo facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:As used herein, the term glucose-6-phosphatase catalytic subunit 1 (G6PC1) refers to catalytic subunit one of the G6PC protein (e.g., having accession number NP_000142.2; SEQ ID NO:1), encoded by the G6PC1 gene (e.g., having accession numbers NM_000151.4 (mRNA; SEQ ID NO:2) and NG_011808.1 (DNA; SEQ ID NO:3)).As used herein, the term glucose-6-phosphatase catalytic subunit 2 (G6PC2) refers to catalytic subunit two of the G6PC protein (e.g., having accession number NP_066999.1 ; SEQ ID NO:4), encoded by the G6PC2 gene (e.g., having accession numbers NM_021176 (mRNA; SEQ ID NO: 5) and NG_011682 (DNA; SEQ ID NO: 6)).As used herein, the term glucose-6-phosphatase catalytic subunit 3 (G6PC3) refers to catalytic subunit one of the G6PC protein (e.g., having accession number NP_612396; SEQ ID NO:7), encoded by the G6PC3 gene (e.g., having accession numbers NM_138387.4 (mRNA; SEQ ID NO:8) and NG_015818 (DNA; SEQ ID NO:9)).As used herein, the term "subject" refers to any animal e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.As used herein, the term "subject suspected of having cancer" refers to a subject that presents one or more symptoms indicative of cancer. A subject suspected of having cancermay also have one or more risk factors. A subject suspected of having cancer has generally not been tested for cancer. However, a "subject suspected of having cancer" encompasses an individual who has received a preliminary diagnosis but for whom a confirmatory test has not been done or for whom the level or severity or grade of cancer is not known.As used herein, the term "subject diagnosed with cancer" refers to a subject who has been tested and found to have cancer. As used herein, the term "initial diagnosis" refers to a test result of initial disease that reveals the presence or absence of disease.As used herein, the term "subject at risk for cancer" refers to a subject with one or more risk factors for developing cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposure, and previous incidents of cancer, preexisting diseases, and lifestyle.As used herein, the term "non-human animals" refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.As used herein, the term "eukaryote" refers to organisms distinguishable from "prokaryotes." It is intended that the term encompass all organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by a nuclear membrane, within which lie the chromosomes, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, the term includes, but is not limited to such organisms as fungi, protozoa, and animals (e.g., humans).As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g. , cancer). Test compounds comprise bothknown and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.As used herein, the term “co-administration” refers to the administration of at least two agent(s) (e.g., polypeptide described herein) or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents / therapies are coadministered, the respective agents / therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents / therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo.As used herein, the term “toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. The peptide or polypeptide may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide”, “oligopeptide,” and “peptide” are used interchangeably herein. The peptide(s)may be produced by recombinant genetic technology or chemical synthesis. The peptide(s) may be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, and size exclusion), or by any other standard techniques known in the art.The recitations “sequence identity,” “percent identity,” “percent homology,” “percent similarity,” or, for example, comprising a “sequence 50% identical to” or “sequence with at least 50% similarity to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, thepercent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.The polypeptide sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the startcodon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include [l-amino acids ( 3 and 2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring- substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D- amino acids, alpha-methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state. For example, the amino acid residue homoSerine (hSer) or homoSerine(Cl) in its unbonded form may take the form of 2- aminobutyric acid (Abu) when participating in an intramolecular bond according to the present invention.For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0039] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single- letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or DF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the polypeptide.In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four-character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N-methylglycine), Aib (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), y-Glu (y-glutamic acid), Gaba (y-aminobutanoic acid), P-Pro (pyrrolidine-3 -carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), hPro (P-homoproline), hPhe (P-homophenylalanine) and Bip ( , diphenylalanine), and Ida (Iminodiacetic acid).The term “pharmaceutically acceptable salt” in the context of the present invention (pharmaceutically acceptable salt of a polypeptide described herein) refers to a salt which is not harmful to a patient or subject to which the salt in question is administered. It may suitably be a salt chosen, e.g., among acid addition salts and basic salts. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the peptides may also be quatemized with alkyl chlorides, bromides, and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like. Other examples of pharmaceutically acceptable salts are described in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977).The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a gene construct) can be introduced into a cell, so as to transform the cell and promote expression (e.g. transcription and translation) of the introduced sequence or knockdown or disruption of the target nucleic. Vectors include, but are not limited to, cells, plasmids, phages, and virusesDETAILED DESCRIPTION OF THE DISCLOSUREProvided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6- phospate (G6PC) (e.g., by administering G6PC polypeptides) for treating cancer.A century ago, Otto Warburg discovered that tumor cells utilized tremendously more glucose and produced large amounts of lactate than normal differentiated cells even in the presence of sufficient oxygen to support mitochondrial oxidative phosphorylation (WarburgO. On the metabolism of cancer cells. Naturwissenschaften. 1924;12: 1131-7). This phenomenon is referred as aerobic glycolysis, and also termed as “the Warburg effect”. All proliferating cells have great bioenergetic and biosynthetic demand to produce new cells, imposing a large requirement for ATP, ribose 5-phosphate, and intermediates that are needed for the synthesis of nucleotides, lipids, and amino acids (Ortega AD, Sanchez-Arago M, Giner-Sanchez D, Sanchez-Cenizo L, Willers I, and Cuezva JM. Glucose avidity of carcinomas. Cancer Lett. 2009;276:125-35; Koppenol WH, Bounds PL, and Dang CV. Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer.2011 ;11 :325-37; Vander Heiden MG, and DeBerardinis RJ. Understanding the Intersections between Metabolism and Cancer Biology. Cell. 2017;168:657-69; Ghergurovich JM, Lang JD, Levin MK, Briones N, Facista SJ, Mueller C, et al. Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer. Med (N Y). 2021 ;2:736-54). Tumor cells exhibit active anabolic metabolism to generate requisite building blocks, and thus, the complete metabolism of glucose to CO and to maximize ATP production through oxidative phosphorylation is against the needs of the proliferating tumor cells. Glucose can be used to generate NADPH, ribose 5-phosphate, and 3 or 6 carbons (glyceraldehyde 3-phosphate or fructose 6-phosphate) via pentose phosphate pathway. While through glycolysis, one molecule glucose will generate 2 molecules of pyruvate, and 2 ATPs. Pyruvate can be converted to acetyl-CoA or oxaloacetate by pyruvate dehydrogenase or pyruvate carboxylase respectively. Acetyl-CoA and oxaloacetate can enter the mitochondrial tricarboxylic acid (TCA) cycle to replenish the intermediates that can be used to make amino acids. Acetyl-CoA can also be used to synthesize the lipids. Therefore, glucose metabolism can provide ATP, NADPH, carbon skeleton for the synthesis of the needed building blocks (nucleotides, lipids, and amino acids) in proliferating tumor cells. However, excessive pyruvate will be converted to lactate to regenerate the NAD+, which is a limited factor for glycolysis. Based on the critical roles of aerobic glycolysis in tumor cells’ proliferation, targeting glycolysis is an attractive therapeutic intervention for tumor treatment (Amer EN, and Rathmell JC. Metabolic programming and immune suppression in the tumor microenvironment. Cancer Cell. 2023 ;41 (3):421 -33; Stine ZE, Schug ZT, Salvino JM, and Dang CV. Targeting cancer metabolism in the era of precision oncology. Nat Rev Drug Discov. 2022;21(2):141-62).In the first step of glycolysis, glucose is required to be phosphorylated to yield glucose 6-phosphate for subsequent reactions. In a reversible reaction, glucose 6-phosphatase catalytic subunit (G6PC) catalyzes the conversion of glucose 6-phosphate to glucose. Severalsolid tumors, including HCC, have low G6PC expression (Tian L, and Liao Y. Identification of G6PC as a potential prognostic biomarker in hepatocellular carcinoma based on bioinformatics analysis. Medicine (Baltimore). 2022;101(33):e29548; Xu WH, Xu Y, Tian X, Anwaier A, Liu WR, Wang J, et al. Large-scale transcriptome profiles reveal robust 20- signatures metabolic prediction models and novel role of G6PC in clear cell renal cell carcinoma. J Cell Mol Med. 2020;24(16):9012-27) and the low G6PC expression is correlated to poor survival rate of these patients (Tian L, and Liao Y. Identification of G6PC as a potential prognostic biomarker in hepatocellular carcinoma based on bioinformatics analysis. Medicine (Baltimore). 2022;101(33):e29548; Xu WH, Xu Y, Tian X, Anwaier A, Liu WR, Wang J, et al. Large-scale transcriptome profiles reveal robust 20-signatures metabolic prediction models and novel role of G6PC in clear cell renal cell carcinoma. J Cell Mol Med. 2020;24(16):9012-27). Moreover, low G6PC expression can promote liver tumor formation through glycogen accumulation (Liu Q, Li I, Zhang W, Xiao C, Zhang S, Nian C, et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell.2021 ; 184(22):5559-76 el9). In this respect, increasing G6PC expression provides a therapeutic strategy for tumor treatment through reduction of the use of glucose. In particular, AMPK can regulate the expression of G6PC (He L, Sabet A, Djedjos S, Miller R, Sun X, Hussain MA, et al. Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell. 2009;137(4):635-46; Wang Y, An H, Liu T, Qin C, Sesaki H, Guo S, et al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep. 2019;29(6): 1511 -23 e5). Experiments described herein investigated the antitumor effects of G6PC in inhibiting glucose utilization in tumor cells.Accordingly, provided herein are G6PC polypeptides and nucleic acids encoding such polypeptides for use in treating cancer.The present disclosure contemplates G6PC1 polypeptides (e.g., those shown in SEQ ID NO:1) or polypeptides with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 1.The present disclosure further contemplates G6PC2 polypeptides ((e.g., those shown in SEQ ID NO:4) or polypeptides with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 4.The present disclosure further contemplates G6PC3 polypeptides ((e.g., those shown in SEQ ID NO:7) or polypeptides with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 7.In some embodiments, polypeptides comprise one or more substitutions relative to SEQ ID NOs: 1, 4, or 7. In some embodiments, polypeptides comprise one or more (e.g., 1, 2,3 4, 5, or more) insertions or additions to the C or N terminus of amino acids to the peptides of SEQ ID NOs: 1, 4, or 7.In some embodiments, the polypeptide comprises, consisting essentially of, or consists of a polypeptide described by SEQ ID NOs: 1, 4, or 7.In some embodiments, polypeptides comprise one or more additional components useful in aiding the polypeptide in entering a cell. For example, in some embodiments, polypeptides are attached to a nanomaterial such as a nanoparticle (See e.g., ((Harish, V. et al. Review on Nanoparticles and Nanostructured Materials: Bioimaging, Biosensing, Drug Delivery, Tissue Engineering, Antimicrobial, and Agro-Food Applications. Nanomaterials (Basel) 12, doi:10.3390 / nanol2030457 (2022); herein incorporated by reference in its entirety).Variants or analogs can differ from the polypeptides described herein by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Current Protocols in Molecular Biology" (Ausubel, 1987). Also included are cyclised peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., 0 or y amino acids.Amino acids include naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamate, and O- phosphoserine, phosphothreonine. An amino acid analog is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium), but that contains some alteration not found in a naturally occurring amino acid (e.g., a modified side chain); the term "amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acid analogs may have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. In one embodiment, an amino acid analog is a D-amino acid, a P-amino acid, or an N-methyl amino acid.Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Non-protein analogs having a chemical structure designed to mimic functional activity of the peptides described herein can be administered according to methods of the disclosure. Variants and analogs may exceed the physiological activity of the original peptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs exhibit the activity of a reference polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. Preferably, the polypeptide or peptide analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.In some embodiments, candidate polypeptides are screened for activity (e.g., using the methods described the experimental section below or another suitable assay).In some embodiments, nucleic acids encoding G6PC are provided. Exemplary, nonlimiting examples of nucleic acids encoding G6PC are described in SEQ ID NO: 2 or 3.In order to express a gene encoding a G6PC polypeptide, one introduces a nucleic acid encoding the gene of interest (e.g., on a plasmid or other vector or as a naked nucleic acid) into a subject. In such instance, a vector suitable for in vivo administration would be utilized, including but not limited to a virus such as retroviruses, adenoviruses, adeno- associated viruses, herpes simplex virus, and the like. See Lundstrom, Viral Vectors in Gene Therapy, Diseases, 2018, 6(2):42. Alternatively, components of the system are administered to a subject via naked polynucleotides (e.g. naked DNA), or physical vehicles such as liposomes and nanoparticles.Vectors may comprise a nucleic acid sequence into which a foreign nucleic acid sequence is inserted. A common way to insert one segment of nucleic acid sequence into another segment of a nucleic acid sequence involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional(foreign) DNA and which can readily be introduced into a suitable cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors which replicate or exist episomally, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.The present disclosure further provides pharmaceutical compositions (e.g., comprising the nucleic acids or polypeptides described above). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of thecompositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the polypeptide is administered in maintenance doses, ranging from 0.1 to 5 mg per kg of body weight, once or more daily, to once every 20 years.Methods of treating cancerThe nucleic acids and polypeptides described herein find use in the treatment cancers. In some embodiments, the agents described herein are administered in combination with one or more additional agents, treatment, or interventions (e.g., agents, treatments, or interventions useful in the treatment of cancer.Examples of anti-cancer therapies include targeting cancer therapy (e.g., targeting the cancer markers described herein), surgery, chemotherapy, radiation therapy, immunotherapy / biological therapy, and photodynamic therapy. Chemotherapeutic agents may also be used for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, anti-metabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes, topoisomerase inhibitors, and cytotoxic antibiotics. Cisplatin, carboplatin, and oxaliplatin are examples of alkylating agents. Other alkylating agents include mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide.Alkylating agents may impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. Alternatively, alkylating agents may chemically modify a cell's DNA.Biological therapy (sometimes called immunotherapy, biotherapy, or biological response modifier (BRM) therapy) uses the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. Biological therapies include interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents. In some embodiments, the biological therapy is immune checkpoint therapy. Immune checkpoint inhibitors target CTLA-4, PD-1, or PD-L1. Examples include but are not limited to, ipilimumab, nivolumab, pembrolizumab, spartalizumab, and atezolizumab.EXPERIMENTALThe following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.Example 1MATERAILS AND METHODSAdenovirusesAdenovirus expression vectors of FLAG-tagged AMPKal-WT, -AMPKal-T172D, and -AMPKal-S496A, -AMPKa2-WT, and -AMPKa2-K45R were generated as described previously (Cao et al., J Biol Chem. 2014;289(30):20435-46 2014). FLAG-tagged human G6pc gene was subcloned into the pENTR2B vector (Invitrogen), and transferred into the pAd / CMV / V5-DEST vector (Invitrogen) by recombination to generate adenoviral expression clones (Cao J, Peng J, An H, He Q, Boronina T, Guo S, et al. Endotoxemia-mediated activation of acetyltransferase P300 impairs insulin signaling in obesity. Nat Commun. 2017;8(l):131).Primary Tumor cells and normal cells and paraffin-embedded sectionsHuman dissociated tumor cells of hepatocellular carcinoma and breast ductal cancer, and human primary hepatocytes were purchased from BioIVT. Human primary mammary epithelial cells were purchased from Lonza Bioscience.Animal experimentsAll animal protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University. The immunodeficient NSG mice were purchased from the Jackson Laboratory. For the assessment of the effect of the peptide on the inhibition of HepG2 xenografts’ growth, 3-millions HepG2 cells and 0.45 nM of NP-Vehicle or NP-G6PC plus VitroGel were subcutaneously implanted into the right flanks of male NSG mice, 0.45 nM of NP-G6PC or NP-control were injected directly into the tumor cells’ implanted sites at day 3, day 6, and day 9, then 0.9 nM of NP-G6PC or NP-control were injected directly into the tumor at day 12, 25, 18, 21, and 25 after implantation. Tumor xenografts were collected 4 weeks after the implantation.Synthesis of peptides and G6PC proteinThe AMPK targeting-peptides (Pa496h and Pa2-491) were synthesized at the Sequencing and Synthesis Facility of Johns Hopkins School of Medicine. G6PC protein was purchased from Creative Biomart.Determination of mitochondrial respiratory activity in tumor cellsHepG2 cells or MCF7 cells were seeded in an XF 96 well plate coated with 0.01% collagen type I. Cells were treated as indicated, followed by the determination of mitochondrial respiratory chain activity using Seahorse XF96 Extracellular Flux Analyzers in Seahorse assay medium (10 mM glucose, 1 mM pyruvate, and 2 mM glutamine, pH7.4). After determination of basal oxygen consumption rates, cells were sequentially treated with oligomycin A (1 pM), FCCP (1 pM), and rotenone (1 pM) along with antimycin A (1 pM). Viable cell numbers were counted and used to normalize the oxygen consumption rate.Determination of cellular glucose levels and cell viabilityA fluorescence labelled 2-deoxy-glucsoe: 2-NBDG (2-(N-(7-nitrobenz-2-oxa-l,3- diazol-4-yl) amino)-2-deoxyglucose) (2-NBDG) (Cat# 72987, Sigma), that cannot undergo glycolysis (Yoshioka K, Takahashi H, Homma T, Saito M, Oh KB, Nemoto Y, et al. A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta. 1996; 1289( 1) :5-9; Bai D, Zhang Y, Shen M, Sun Y, Xia Q, Zhang Y, et al. Hyperglycemia and hyperlipidemia blunts the Insulin-Inpp5f negative feedback loop in the diabetic heart. Sci Rep. 2016; 6:22068), was used as an indicator of cellular glucose concentrations. After the treatment of cells, medium was changed to glucose-free DMEM containing 200 LI M of fluorescence labelled 2-NBDG for 20 min, then washed with no color DMEM, and the fluorescence intensities were determined using Zeiss confocal microscope (Zeiss Confocal LSM 880). Cell viability was determined using the RealTime- Glo MT Cell Viability Assay (Promega) using procedure recommended by the manufacturer.Generation of G6PC nanoparticlesNanoparticles were generated using an established procedure of a sequential flash nanocompl ex ati on (FNC) and flash nanoprecipitation (FNP) process to co-encapsulate peptide or protein through complexation with negatively charged dextran sulfate within poly (ethylene glycol )- / ?-poly (lactic acid-co-glycolic acid) (PEG-&-PLGA) nanoparticles. A four- inlet multi-inlet vortex (MIV) mixer and a 3-inlet confined impinging jet (CIJ) mixer were connected in tandem to accommodate the FNC / FNP process (Hanwright et al., Biomaterials. 2022;280: 121244 2022; Hu et al., 2019; Zhu et al., 2022).RNA-seq analysis.R A samples were extracted using TRlzol reagent (ambion). R easy Micro Kit (Qiagen) was used to clean the RNA samples. RNA-seq analysis was conducted in the Johns Hopkins Deep Sequencing and Microarray Core Facility.Statistical analysesStatistical significance was calculated with a Student’s t test and ANOVA test. Significance was accepted at the level of p < 0.05. At least 3 samples per group were chosen for statistically meaningful interpretation of results and differences in the studies using the Student’s t test and analysis of variation.ResultsInduction of glucose-6-phosphatase by targeting-peptides reduces cellular glucose levelsIt was assessed whether targeting-peptides could affect the gene expression in tumor cells, by treating HepG2 cells with Pa496h or Pa2-491 (See e.g., PCT / US2023 / 071 191; herein incorporated by reference in its entirety), and conducting RNA-seq analysis. Principal component analysis (PC A) demonstrated that Pa496h and Pa2-491 treatment led to similar marked changes in the transcriptomes (Figure la), but differences were also noticed (Figure lb). Both Pa496h and Pa2-491 drastically increased the mRNA levels of the rate-limitinggluconeogenic glucose-6-phosphatase (G6pc or G6pcl ) by 10-fold (Figures 1c, d). Quantitative PCR confirmed the augmentation of the mRNA levels of G6pc by Pa496h and Pa2-491 in HepG2 cells (Figure le). Furthermore, the augmentation of G6pc mRNA levels is also accompanied by elevated the protein levels of G6PC in HepG2 cells and MCF7 cells (Figures If, g).Considering the importance of G6PC in glucose metabolism, it was contemplated that increased G6PC by these targeting-peptides would catalyze the conversion of glucose 6- phosphate to glucose, leading to the inhibition of glucose utilization in tumor cells. Indeed, Pa496h significantly reduced the oxygen consumption rate (OCR) and extracellular acidification rate in the glycolytic assay medium (Figures Ih, i), which reflects decreased glycolytic flux and lactate production in tumor cells by Pa496h (Divakaruni AS, Paradyse A, Ferrick DA, Murphy AN, and Jastroch M. Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol. 2014;547:309-54). After the removal of phosphate group from glucose 6-phosphate by increased G6PC, glucose is released from the cells because of the bidirectional function of glucose transporters. To test this, assays were conducted to examine cellular glucose levels. In these experiments, a fluorescence labelled 2-deoxy-glucsoe: 2-NBDG (2-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl) amino)-2- deoxyglucose), that cannot undergo glycolysis (Yoshioka K, Takahashi H, Homma T, Saito M, Oh KB, Nemoto Y, et al. A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta. 1996; 1289(1 ): 5-9; Bai D, Zhang Y, Shen M, Sun Y, Xia Q, Zhang Y, et al. Hyperglycemia and hyperlipidemia blunts the Insulin-Inpp5f negative feedback loop in the diabetic heart. Sci Rep. 2016;6:22068), was used as an indicator of cellular glucose concentrations. Treatment with Pa496h significantly reduced 2-NBDG levels in HepG2 cells and MCF7 cells (Figures Ij, k). Consistently, Pa496h treatment significantly reduced 2-NBDG levels in hepatocellular carcinoma-dissociated tumor cells (HCC-DTCs) and breast cancer DTCs (Figures 11, m). In contrast, Pa496h treatment significantly elevated 2-NBDG levels in primary hepatocytes prepared from elderly patient (Figure In) because Pa496h could decrease the expression of G6pc gene to suppress glucose production in liver hepatocytes (Pearah A, Ramatchandirin, B., Namachivayam, K., Liu, T., Radovick, S., Sesaki, H., Wondisford, F.E., O’Rourke, A., He, L. Blocking AMPKalphaS496 phosphorylation improves mitochondrial dynamics and hyperglycemia in aging and obesity. Cell Chem Biol 2023; 30: 585-1600 el586). Similarly, Pa496h treatment elevated 2-NBDG levels in primary human mammary epithelial cells (HMEC) prepared from normal subject (Figure lo).G6pc overexpression inhibits the growth of tumor cellsPrevious studies showed that low G6pc expression is associated with poor survival in patients with HCC and renal cell carcinoma (Tian L, and Liao Y. Identification of G6PC as a potential prognostic biomarker in hepatocellular carcinoma based on bioinformatics analysis. Medicine (Baltimore). 2022;101(33):e29548; Xu WH, Xu Y, Tian X, Anwaier A, Liu WR, Wang J, et al. Large-scale transcriptome profiles reveal robust 20-signatures metabolic prediction models and novel role of G6PC in clear cell renal cell carcinoma. J Cell Mol Med. 2020;24(l 6) :9012-27), and low G6pc expression can promote liver tumor formation (Liu Q, Li J, Zhang W, Xiao C, Zhang S, Nian C, et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell. 2021;184(22):5559-76 el9). To assess whether elevation of G6PC could inhibit the growth of tumor cells, an adenoviral expression vector of G6pc gene was generated (Figure 2a), and G6PC was overexpressed in hepatoma HepG2 cells or MCF7 cells, and it was found that overexpression of G6PC significantly reduced 2-NBDG levels in both HepG2 cells or MCF7 cells compared to the 2-NBDG levels HepG2 cells or MCF7 cells with expression of GFP (Figures 2b, c). Overexpression of G6pc in HepG2 cells or MCF7 cells decreased their cell viability by 34.2 and 36.8%, respectively (Figures 2d, e). Overexpression of G6pc in HCC-DTCs inhibited the growth of tumor cells in a timedependent manner and decreased the cell viability byand 91.1% at 16 h, 48 h and 64 h after the adenoviral transduction (Figure 2f). A similar pattern of decreased cell viability of the breast cancer DTCs was observed after the addition of adenoviral G6pc expression vector, and cell viability decreased by 20.0%, 70.3%, and 84.8% at 16 h, 48 h and 64 h after the adenoviral transduction (Figure 2g). However, 40 h after the addition of adenoviral G6pc expression vector, cell viability was not significantly affected in primary hepatocytes (Figure 2h). In primary normal HMEC, 16 h after the addition of G6pc adenovirus, cell viability was not significantly affected (Figure 2i), and 40 h after the addition of G6pc adenovirus, a 29.9% decrease in cell viability of HMEC (Figure 2j) compared to 84.8% decrease in cell viability of breast cancer DTCs was observed (Figure 2g).In addition, overexpression of G6PC in breast cancer JIMT1 cells, small cell lung can H69PR cells, B-lymphoma Ramos cells, melanoma HT144 cells, pancreatic adenocarcinoma BxPC3 cells, and primary glioblastoma cells also significantly decreased their cell viability (Figures 2k-p).Inhibition of tumor cells’ growth by G6PC nanoparticlesIt was contemplated that increased G6PC levels would lead to the reduction of glucose utilization. G6PC nanoparticles were used to treat HepG2 cells for 6 h. Treatment with G6PC nanoparticles decreased oxygen consumption rate when glucose was provided as the main substrate, and this treatment significantly decreased basal respiration, ATP-linked respiration, and non-mitochondrial respiration (Figure 3a, b). In MCF7 cells, treatment with G6PC nanoparticles decreased oxygen consumption rate, and significantly decreased basal respiration, ATP-linked respiration, and maximal respiration capacity (Figure 3c, d). These results revealed that elevated G6PC can decrease glucose utilization. Furthermore, treatment with G6PC nanoparticles reduced the cell viability of HepG2, MCF7, B-lymphoma Ramos cells, human kidney tumorigenic Hek293 cells, HCC-DTCs, and breast cancer DTCs by 93.9%, 84.9%, 73.8%, 92.2%, 61.6%, and 52.4%, respectively (Figures 3e-j). In comparison, treatment with G6PC nanoparticles had no significant impact on the growth of human primary hepatocytes (Figure 3k) and had a mild effect on the growth of human normal HMEC (Figure 31). HepG2 cells were subcutaneously injected together with NP-G6PC in the immunodeficient NSG mice, followed by NP-G6PC treatment every 3-day for a period of 4 weeks after the implantation. Treatment with NP-G6PC significantly reduced tumor xenografts’ volume and weight (Figures 3m-o). These data confirmed that elevated G6PC can inhibit the growth of tumor cells, but has no or mild effect on the growth of normal cells.Induction of G6pc by targeting-peptide is through aPKCi / 1 degradationPrevious studies documented that activation of AMPK leads to the suppression of G6pc expression and glucose production in the liver hepatocytes through the phosphorylation of CBP at S436 and disassembly of the CREB-CBP-CRTC2 complex (gluconeogenic engine) (Cao, J., Meng, S., Chang, E., Beckwith-Fickas, K., et al. Low concentrations of metformin suppress glucose production in hepatocytes through AMP-activated protein kinase. J Biol Chem 2012; 289: 20435-20446; He, L., Sabet, A., Djedjos, S., Miller, R., et al. Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell 2009; 137: 635-646). Treatment with targeting-peptide Pa496h decreased the mRNA and protein levels of G6pc in primary hepatocytes and liver of obese mice (Pearah A, Ramatchandirin, B., Namachivayam, K., Liu, T., Radovick, S., Sesaki, H., Wondisford, F.E., O’Rourke, A., He, L. Blocking AMPKalphaS496 phosphorylation improves mitochondrial dynamics and hyperglycemia in aging and obesity. Cell Chem Biol 2023; 30: 585-1600 el 586). However, in tumor cells, activation of AMPK by Pa496 or Pa2-491 led to drastically increased mRNA and protein levels of G6pc (Figures Ic-g), that is the opposite resultsobserved in liver hepatocytes, indicating that activation of AMPK by Pa496 or Pa2-491 may not result in the phosphorylation of CBP at S436 and CRTC2 at S 171 and disassembly of the CREB-CBP-CRTC2 complex.In agreement with previous reports that activation of AMPK can increase the phosphorylation of CREB at S133 (Thomson, D. M., Herway, S. T., Fillmore, N., Kim, H., Brown, J. D., Barrow, J. R., and Winder, W. W. (2008) AMP-activated protein kinase phosphorylates transcription factors of the CREB family. J Appl Physiol 1985; 104: 429- 438), Pa496h treatment increased the phosphorylation of CREB at SI 33 (Figures If). CREB is constitutively phosphorylated at SI 33 (Koo SH, Flechner L, Qi L, Zhang X, Screaton RA, Jeffries S, et al. The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism. Nature. 2005;437(7062): 1109-11), indicating that the gluconeogenic engine, CREB-CBP-CRTC2 complex, may not be affected by the targeting-pep tides, even though with AMPK activation. Corroborating to this notion, Pa496h treatment was unable to change the phosphorylation of CBP at S436 in HepG2 cells (Figures If). Moreover, Pa496h treatment led to decreased protein levels of aPKCi / 1 (Figures If, 3p, q), this is through increasing the ubiquitination and degradation of aPKCi / 1, an oncogene in various forms of human cancer (Ishiguro H, Akimoto K, Nagashima Y, Kojima Y, Sasaki T, Ishiguro- Imagawa Y, et al. aPKClambda / iota promotes growth of prostate cancer cells in an autocrine manner through transcriptional activation of interleukin- 6. Proc Natl Acad Sci U S A. 2009; 106(38): 16369-74; Phillips E, Lang V, Bohlen J, Bethke F, Puccio L, Tichy D, et al. Targeting atypical protein kinase C iota reduces viability in glioblastoma stem-like cells via a notch signaling mechanism. Int J Cancer. 2016;139(8):1776-87), in HepG2 and MCF7 cells (Figure 3p, q). Therefore, in tumor cells, the targeting-peptides can stimulate the G6pc expression by augmenting CREB phosphorylation at SI 33 to activate the gluconeogenic engine CREB-CBP-CRTC2 complex.All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMSWe claim:

1. A method of treating cancer in a subject, comprising: administering a composition comprising glucose-6-phosphatase to said subject.

2. The method of claim 1, wherein said glucose-6-phosphatase is glucose-6- phosphatase catalytic subunit 1.

3. The method of claim 1 or 2, wherein said glucose-6-phosphatase is a glucose- 6-photphatase polypeptide.

4. The method of claim 1 or 2, wherein said glucose-6-phosphatase is a nucleic acid encoding a glucose-6-photphatase polypeptide.

5. The method of any of the preceding claims, wherein said cancer is selected from the group consisting of breast cancer, liver cancer, melanoma, pancreatic cancer, lung cancer, brain cancer, and lymphoma.

6. The method of any of the preceding claims, wherein said composition is a pharmaceutical composition.

7. The method of any of the preceding claims, wherein said composition is administered to the site of said cancer.

8. The method of any of the preceding claims, wherein said composition is administered systematically.

9. The method of any of the preceding claims, wherein said glucose-6- phosphatase inhibits the growth of or kills tumor cells but not normal cells.

10. The use of a composition comprising glucose-6-phosphatase to treat cancer in a subject.

11. A composition comprising glucose-6-phosphatase for use in treating cancer in a subject.

12. The composition of claim 11, wherein said composition is a pharmaceutical composition.

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