CHRM3 as a marker and target for colon cancer therapy

Abstract

The present invention relates to a method for predicting the prognosis of a subject suffering from a disease. The present invention further relates to a diagnostic kit for predicting said prognosis. The present invention further relates to the use of CHRM3 mRNA or protein expression as a marker for said prognosis. The present invention further relates to a method for treating a subject suffering from a disease or disorder associated with CHRM3 mRNA or protein expression and CHRM3 protein activity.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting the prognosis of a subject suffering from a disease. The present invention further relates to a diagnostic kit for predicting said prognosis. The present invention further relates to the use of CHRM3 mRNA or protein expression as a marker for said prognosis. The present invention further relates to a method for treating a subject suffering from a disease or disorder associated with CHRM3 mRNA or protein expression and CHRM3 protein activity. BACKGROUND OF THE INVENTION

[0002] Cholinergic receptors are divided into two major classes, nicotinic acetylcholine receptors and muscarinic acetylcholine receptors, based on their responsiveness to nicotine and muscarine. Unlike nicotinic acetylcholine receptors, which are ion channels, muscarinic acetylcholine receptors belong to the superfamily of G protein-coupled receptors that activate ion channels via second-messenger cascades. Muscarinic acetylcholine receptors, also known as cholinergic / acetylcholine receptor M3 or muscarinic 3, are encoded by the human gene CHRM3. Muscarinic acetylcholine receptors are divided into subtypes M1–M5 based on their cellular actions, pharmacology, and molecular biology. They fulfill several roles, including serving as the primary terminal receptors stimulated by acetylcholine released from postganglionic fibers in the parasympathetic nervous system.

[0003] Among the several subtypes of muscarinic acetylcholine receptors, the M3 muscarinic receptor subtype is present in many parts of the body, including vascular smooth muscle and the lungs. This M3 receptor is Gq-coupled and mediates an increase in intracellular calcium, which typically causes smooth muscle contraction, such as that seen during bronchoconstriction and bladder voiding. However, in the vasculature, activation of M3 in vascular endothelial cells increases the synthesis of nitric oxide, which then diffuses to adjacent vascular smooth muscle cells, relaxing them, thereby explaining the paradoxical effects of parasympathomimetics on vascular tone and bronchiolar tone. The M3 receptor is also present in many glands, such as the salivary glands, which help stimulate secretion. Furthermore, the M3 receptor is a G-protein of the Gq class that increases phospholipase C.

[0004] Furthermore, activation of the M3 subtype muscarinic receptor is known to play an important role in promoting the proliferation of colon cancer cells.

[0005] Cheng et al., Oncotarget, 2017, Vol. 8, (No. 13), 21106, reported that activation of the M3 muscarinic receptor (M3R) promotes the proliferation, migration, and invasion of colon cancer cells. The expression of M3R was compared in normal colon, normal colon with colon adenoma, and cancer-affected colon with primary and metastatic tumors (colon cancer). It was concluded that M3R expression plays an important role in the early progression and invasion of colon tumors, but is less important once the tumor has already spread.

[0006] Ali et al., Int. J. Mol. Sci. 2021, 22,716, described that most colorectal cancers overexpress M3 muscarinic receptors (M3R). Furthermore, activation of these receptors has been shown to stimulate cellular programs that contribute to the proliferation, survival, and spread of these cancers. In vivo studies in mouse models have demonstrated that inhibiting M3R expression or activation attenuates the development and progression of colon cancer. Ali et al. are investigating potential therapies targeting muscarinic receptor expression, activation, and signaling in colorectal cancer, including the development of muscarinic receptor antagonists, targeting matrix metalloproteinases, M3R-stimulated EGFR transactivation, or interference with RAS, BRAF, and mitogen-activated protein kinase (MAPK) signaling components downstream of M3R and EGFR.

[0007] Cheng et al., Molecular Cancer 2014, 13,77, describe the identification of a novel gene, Zfp277, whose expression pattern coincides with mediating the differential effects of CHRM3 and CHRM1 gene ablation on intestinal tumors in mice.

[0008] Tolaymat et al., Cancers 2019, 11,308, described that M3 muscarinic receptor (M3R) mRNA and protein are overexpressed in colon cancer. M3R is activated by both conventional muscarinic ligands (e.g., acetylcholine) and non-conventional muscarinic ligands (e.g., bile acids). This paper focuses on the role of key protein kinases downstream of M3R activation in promoting colon cancer progression and spread.

[0009] Goto et al., Oncogene 39, 2020, 4014, described that muscarinic receptors promote castration-resistant growth of prostate cancer. Activation of muscarinic acetylcholine receptors was studied.

[0010] Ashizawa et al., Hepatology Commun 3, 2019, 954, reported that olfactomedin 4 (OLFM4) is induced by the LGR5-Wnt signaling pathway and is associated with malignant tumor progression and poor prognosis in hepatocellular carcinoma (HCC) by regulating STAT3-induced tumor cell proliferation and cancer stem cell-like properties. OLFM4 may be a prognostic predictor.

[0011] Fumagalli A. et al., Cell Stem Cell, 26, 2020, 569, states that most metastases of colorectal cancer are caused by Lgr5 cells.

[0012] Felton J. et al., Curr Mol. Pharmacol. 2018, 11(3), 184-190, describes the role of M3R in colon cancer.

[0013] Bernat-Peguera A. et al., Clin. Cancer Res., 27, 2021, 1491, analyzed the molecular characteristics underlying the response to EGFR inhibitors and the underlying mechanisms of cutaneous squamous cell carcinoma. It was suggested that combination therapy targeting EGFR and FGFR could be used to treat cutaneous squamous cell carcinoma.

[0014] Calaf GM et al., Cancer 14, 2022, 2322, 1, report on the correlation between growth factors and M3 receptors, differences in survival adjusted for clinical staging, and the relationship between gene expression and immune infiltration levels in various human cancers.

[0015] Houghton LA et al., Aliment Pharm. Therap., 1997, 11, 561 discloses that zamifenacin can act as an M3 selective muscarinic antagonist to reduce colonic motor activity in patients with irritable bowel syndrome.

[0016] Steers WD et al., Urol. Clin. N. Am., 2006, 33,475, describes darifenacin as an antimuscarinic agent for treating overactive bladder and urge urinary incontinence.

[0017] US2017 / 0246267 describes a method for treating gastric or colon cancer in a subject by administering a cholinergic antagonist, a botulinum toxin, an NGF inhibitor, a TRK inhibitor, or performing surgical denervation. This document does not provide a hint to those skilled in the art that the gene expression of M3 and additional genes (related genes) forms a prognostic signature that can predict a patient's prognosis. Furthermore, this document does not provide a hint that a therapeutic signature can be formed based on the finding that sensitivity to treatment with an M3 inhibitor or a combination of an M3 inhibitor and another inhibitor depends on the expression and / or coexpression of M3-responsive (or active) genes.

[0018] Yahui et al., American J. Translation Research, 2015, pp. 902-911, are concerned with the study of CHRM3 as a prognostic tool for endometrial cancer, particularly in combination with clinicopathological risk factors such as vascular invasion and lymphatic metastasis. Furthermore, it is disclosed that the M3 receptor can be used for tumor therapy of endometrial cancer.

[0019] Xiaodong et al., World Neurosurgery, vol. 167, 2022, studied the prognostic role of genes derived from genomic instability in glioblastoma (GBM). The risk score was based on nine genes, including CHRM3.

[0020] Novus Biologicals (EPW20220840303), p 1-4, 2019, is a datasheet for a kit containing muscarinic acetylcholine receptor M3 / CHRM3 antibodies.

[0021] CN 103393628 discloses the use of muscarinic receptor 3 as a drug target in the treatment of prostate cancer. Muscarinic receptor 3 receptor antagonists are said to inhibit the proliferation and migration of prostate tumor cells. Darifenacin is mentioned as a potential antagonist.

[0022] It is an object of the present invention to provide a method for predicting the prognosis of a subject suffering from a disease or disorder based on CHRM3 expression.

[0023] In particular, it is an object of the present invention to provide a method for predicting whether a patient suffering from cancer will have a poor or good prognosis, particularly for patients suffering from cancer of epithelial origin, in particular colorectal cancer.

[0024] It is a further object of the present invention to provide methods for treating a subject suffering from a disease or disorder based on CHRM3 mRNA overexpression and CHRM3 protein activity, particularly to select for treatment patients with an M3-responsive characteristic and exclude patients without the characteristic.

[0025] The method according to the invention has the following advantages: - The method helps patients decide whether to pursue further therapeutic measures and / or monitoring as an alternative to non-intervention. - The Act allows for the selection of different patient populations, for example, patients who do not require further treatment and / or monitoring, patients who may benefit from further treatment and / or monitoring, patients who should receive further treatment or palliative treatment taking into account decisions made in accordance with the guidelines, or patients who can be spared such treatment. In tumors with elevated CHRM3 levels, CHRM3 activity can be assessed to determine whether to treat the subject with a CHRM3 inhibitor. CHRM3 activity can be measured from changes in gene expression levels in response to CHRM3 stimulation. Summary of the Invention

[0026] The present invention relates to a method for predicting the prognosis of a subject suffering from a disease or disorder based on the expression level of CHRM3, the method comprising the steps of: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein with a control level; Here, a significant increase in the expression level of CHRM3 mRNA or CHRM3 protein compared to the control level indicates that the subject is likely to have a poor prognosis for the disease.

[0027] In particular, the present invention relates to a method for predicting the prognosis of a subject suffering from colorectal cancer based on the expression level of CHRM3, the method comprising the steps of: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein with a control level; Here, a significant increase in the expression level of CHRM3 mRNA or CHRM3 protein compared to the control level indicates that the subject is likely to have a poor prognosis for the disease.

[0028] The present invention further relates to a kit for predicting the prognosis of a subject suffering from a disease or disorder based on the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one further gene associated with CHRM3 mRNA expression, preferably a gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1, the kit comprising means for measuring the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one gene associated therewith.

[0029] In particular, the present invention relates to a kit for predicting the prognosis of a subject suffering from colorectal cancer based on the expression level of CHRM3 mRNA or CHRM3 protein, and optionally at least one further gene associated with CHRM3 mRNA expression, preferably a gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1, the kit comprising means for measuring the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one gene associated therewith.

[0030] The present invention further relates to the use of CHRM3 mRNA or CHRM3 protein expression as a marker for the prognosis of a subject suffering from a disease or disorder, optionally by using at least one additional gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1.

[0031] In particular, the present invention further relates to the use of CHRM3 mRNA or CHRM3 protein expression as a marker for the prognosis of subjects suffering from colorectal cancer, optionally by using at least one further gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1.

[0032] The present invention further relates to a method for treating a subject suffering from a disease or disorder associated with overexpression of CHRM3 mRNA or CHRM3 protein, the method comprising the steps of: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein with a control level; If the expression level of the CHRM3 mRNA or CHRM3 protein is significantly elevated compared to the control level, the subject is subjected to anti-tumor treatment.

[0033] In particular, the present invention also relates to a method for treating a subject suffering from colorectal cancer associated with overexpression of CHRM3 mRNA or CHRM3 protein, comprising the steps of: a) obtaining a sample from a tumor of a subject with colorectal cancer; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein with a control level; If the expression level of the CHRM3 mRNA or CHRM3 protein is significantly elevated compared to the control level, the subject is subjected to anti-tumor treatment.

[0034] The present invention further relates to a method for treating a subject suffering from a disease or disorder associated with overexpression of CHRM3 mRNA or CHRM3 protein and activity of CHRM3 protein, the method comprising the steps of: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein and the activity of CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein and the activity of the CHRM3 protein with a control level; If the expression level of CHRM3 mRNA or CHRM3 protein and the activity of CHRM3 protein are significantly elevated compared to the control levels, the subject is treated with an effective amount of an agent that inhibits CHRM3 activity.

[0035] In particular, the present invention also relates to a method for treating a subject suffering from colorectal cancer associated with overexpression of CHRM3 mRNA or CHRM3 protein and activity of CHRM3 protein, comprising the steps of: a) obtaining a sample from a tumor of a subject with colorectal cancer; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein and the activity of CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein and the activity of the CHRM3 protein with a control level; If the expression level of CHRM3 mRNA or CHRM3 protein and the activity of CHRM3 protein are significantly elevated compared to the control levels, the subject is treated with an effective amount of an agent that inhibits CHRM3 activity.

[0036] "Protein" is used interchangeably with polypeptide and includes fragments and domains of proteins as well as whole proteins.

[0037] In the sense of the present invention, the term "subject" refers to any human or animal. Animals (non-human) include all vertebrates, e.g., mammals and non-mammals, including cows, sheep, pigs, goats, horses, poultry, dogs, cats, non-human primates, rodents, etc. In one embodiment, the subject is a human subject.

[0038] As used herein, the term "marker" refers to a gene whose gene product is used to measure expression levels.

[0039] The gene expression of M3 (CHRM3 mRNA or protein expression) and additional genes (so-called "associated genes") form a prognostic signature (fingerprint) that can predict patient outcome. This prognostic signature can be used to establish a prognostic rating scale.

[0040] The expression of M3 (CHRM3 mRNA or protein expression) and additional genes (so-called "response genes") forms a therapeutic signature that characterizes a subject's tumor sensitivity to treatment, particularly to treatment with an M3 inhibitor or a combination of an M3 inhibitor with another inhibitor. This therapeutic signature can be used to establish a therapeutic assessment scale.

[0041] In other words, the associated gene is selected from a group of genes whose expression is associated with CHRM3 mRNA or CHRM3 protein expression. The responsive gene is selected from a group of genes that are regulated by CHRM3 protein activity. The associated gene and the responsive gene are selected from a group of genes, but are not necessarily the same.

[0042] Related genes can be obtained by measuring the expression of genes in cancer biopsies or patient samples. In patients with high or low CHRM3 expression, the expression levels of CHRM3-related genes are used to weight the CHRM3 expression level.

[0043] Response genes are identified by experimentally inhibiting CHRM3 in CHRM3-expressing human cancer cells and measuring changes in gene expression upon CHRM3 inhibition. To this end, cancer cells are cultured and CHRM3 activity is inhibited. Inhibition is achieved by using small molecule inhibitors of CHRM3 (e.g., darifenacin or zamifenacin) or by knocking down CHRM3 mRNA, which depletes CHRM3 protein from the cells. Cancer cells are harvested before and at various time points after CHRM3 inhibition. Typical time points are 1, 2, 3, 4, 5, 6, or 7 days. In harvested cancer cells, gene expression is measured by RNA sequencing, generating a transcriptome-wide readout. The transcriptome-wide expression after CHRM3 inhibition is compared to control levels, and gene sets are identified using various established methods. Changes in gene expression after CHRM3 inhibition reveal which genes exhibit CHRM3 activity. These genes constitute a signature of response genes.

[0044] CHRM3 expression is defined by the level of CHRM3 mRNA or protein detected in a sample. CHRM3 mRNA levels can be measured by quantitative RT-PCR, in situ hybridization, or RNA sequencing. CHRM3 protein expression levels can be measured using antibodies, immunohistochemistry, ELISA assays, or mass spectrometry.

[0045] CHRM3 activity is defined as an active state caused by ligand binding and / or overexpression leading to the expression of CHRM3-responsive genes and proteins, and can be detected by quantitative RT-PCR or RNA sequencing.

[0046] In the sense of the present invention, the term "prognosis" is the outcome of a treatment or course of treatment for a particular disease.

[0047] The prognosis is measured based on one or a set of criteria at a specific time point during or after treatment. Considering prognosis helps determine the effectiveness and appropriateness of medical intervention and evaluate it compared to alternatives, especially no intervention.

[0048] Disease prognosis has various definitions, for example, it is defined using various endpoints. One way to measure prognosis is "long-term" survival, which refers to survival for a certain period of time, for example, at least 3 years, after diagnosis and / or initial treatment. Another way is "recurrence-free survival" (RFS), which refers to survival from diagnosis and / or initial treatment to cancer recurrence or death from cancer recurrence (usually in years). Another way to measure it is "overall survival" (OS), which refers to survival from diagnosis and / or initial treatment to death from any cause (usually in years). Yet another way is "disease-free survival" (DFS), which refers to survival from diagnosis and / or initial treatment to first cancer recurrence or death from any cause (usually in years).

[0049] Because "prognostic analysis" often focuses on changes in quality of life, each preventive or therapeutic measure can be evaluated in a way that is more meaningful to future subjects than is possible with so-called surrogate markers, parameters, or endpoints (these are measurable variables that are not directly related to the person involved, such as measurements, laboratory values, or tumor diameter).

[0050] Instead of surrogate parameters or intuitive descriptions of cases (e.g., "cured" / "not cured"), the overall description of the situation needs to be defined as precisely as possible.

[0051] Fibroblast growth factors (FGFs) are proteins that stimulate cell growth and differentiation. FGF receptors (FGFRs) bind to FGFs, forming protein-ligand interactions.

[0052] Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation. The EGF receptor (EGFR) binds to EGF, forming a protein-ligand interaction.

[0053] Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), also known as G protein-coupled receptor 49 (GPR49) or G protein-coupled receptor 67 (GPR67), is a protein encoded by the LGR5 gene in humans.

[0054] Olfactomedin 4 is a protein that in humans is encoded by the OLFM4 gene.

[0055] TNF receptor-associated factor 5 is a protein that in humans is encoded by the TRAF5 gene.

[0056] The CD46 complement regulatory protein, also known as CD46 (cluster of differentiation 46) and membrane cofactor protein, is a protein that in humans is encoded by the CD46 gene.

[0057] Transforming growth factor beta 1 or TGF-β1 is a polypeptide member of the transforming growth factor beta superfamily of cytokines. In humans, TGF-β1 is encoded by the TGFB1 gene.

[0058] Sodium / glucose cotransporter 1 (SGLT1) is a protein in humans that is encoded by the SLC5A1 gene, which codes for the production of the SGLT1 protein lining the absorptive cells of the small intestine and the epithelial cells of the renal tubules of the nephron, for the uptake of glucose into the cells.

[0059] 2-oxoglutarate receptor 1 (OXGR1), also known as cysteinyl leukotriene receptor E (CysLTE) and GPR99, is a protein encoded by the OXGR1 gene in humans.

[0060] The first embodiment relates to a method for predicting the prognosis of a subject suffering from a disease or disorder based on the expression level of CHRM3 mRNA.

[0061] Step a) In step a), a sample is taken from a tumor of a subject with a cancer of epithelial origin, in particular colorectal cancer.

[0062] Suitable samples include tissues or cells from tumors of subjects with cancers of epithelial cell origin. Preferably, the samples include biopsy samples such as tumor biopsies, primary tissues, and metastatic tissues. In particular, the specimens can be obtained by needle biopsy, image-guided biopsy, surgical (excision) biopsy, shave / punch biopsy, endoscopic biopsy, laparoscopic biopsy, and combinations thereof.

[0063] The term "taking a sample from a tumor" as used herein refers to a sample obtained from a patient for diagnostic purposes. This tumor sample can be obtained from a patient by conventional means known to those skilled in the art, i.e., by biopsy (taken by aspiration or puncture, excision, or other surgical method leading to biopsy or excised cellular material). In this way, it is possible to obtain tissue for the method of the present invention. However, the tumor will not be (completely) removed during sample collection. In the sense of the present invention, "taking a sample from a tumor" is not intended to refer to a therapeutic treatment method.

[0064] Step b) and Step c) In step b) of the method of the invention, the expression level of CHRM3 mRNA or protein is measured.

[0065] The expression levels of said proteins can be measured by targeted mass spectrometry, especially on tumor tissue.

[0066] The expression level of the CHRM3 protein can be measured using antibodies or other suitable techniques such as immunohistochemistry, ELISA assays, and mass spectrometry. The level of the CHRM3 mRNA can be measured by quantitative RT-PCR, in situ hybridization, or RNA sequencing.

[0067] Antibodies for use in the present invention can be produced by any suitable method known in the art, including, but not limited to, polyclonal, monoclonal, humanized, phage display-derived, or chimeric antibodies.

[0068] In step c) of the method of the invention, the expression level of CHRM3 mRNA or CHRM3 protein is compared with a control level.

[0069] Thus, a significant increase in the expression level of CHRM3 mRNA or CHRM3 protein compared to the control level indicates a high likelihood of a poor prognosis for the disease in the subject. In particular, the expression level of CHRM3 mRNA or CHRM3 protein, as defined above and below, can be used in a rating scale (prognostic rating scale) used to determine whether a subject is likely to have a poor prognosis and should be treated and / or monitored.

[0070] Said control level refers to the expression level of protein in non-cancer tissue.In other words, said control level refers to the expression level of protein in normal non-tumor tissue.This refers to the normalized level of gene product, for example, the normalized value measured for the RNA expression level of gene or the polypeptide expression level of gene.

[0071] An expression level that is "significantly elevated" (relative to a control level) is one that is statistically significant different from the control level, using suitable statistical methods well known in the art.

[0072] Methods for measuring the expression level are conventional and routine. Typically, this measurement relies on the existence of a set of antibodies specific for the overexpression of the protein, for example. Such antibodies are commercially available or can be routinely generated using conventional procedures.

[0073] Assays for measuring activity and / or expression levels can be readily adapted to a high throughput format, for example, using robotics where appropriate.

[0074] Preferably, steps b) and c) further comprise measuring the expression level of at least one gene associated with CHRM3 mRNA expression and comparing the expression level of at least one gene associated with CHRM3 mRNA expression with a control level.

[0075] In particular, the gene associated with CHRM3 mRNA expression is selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1.

[0076] Thus, a significantly elevated expression level of at least one gene associated with CHRM3 mRNA or CHRM3 protein expression compared to control levels is a further indication that the subject is likely to have a poor prognosis for the disease. In particular, the expression level of at least one associated gene can be further used in a rating scale (prognostic rating scale), as defined below, used to determine whether the subject is likely to have a poor prognosis and should be treated and / or monitored.

[0077] Preferably, the expression levels of CHRM3 mRNA and at least one further associated gene are converted into a rating scale (prognostic rating scale).

[0078] Advantageously, the method of the present invention allows for the establishment of a rating scale based on the expression levels of CHRM3 mRNA and / or CHRM3 protein and, optionally, at least one additional associated gene. Advantageously, this rating scale can be used to determine whether the subject is likely to have a poor prognosis. In particular, the expression levels of CHRM3 mRNA and / or CHRM3 protein and, optionally, at least one additional associated gene can be used to determine whether the subject should be treated with an anti-tumor therapy and / or monitored for the progression of the disease. This method allows for the determination of disease at a very early stage, when markers used in state-of-the-art staging protocols (such as other tumor markers or the occurrence of metastases) usually cannot yet be detected.

[0079] In a second embodiment, the present invention relates to a kit for predicting the prognosis of a subject suffering from a disease or disorder, in particular colorectal cancer, based on the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one further gene associated with CHRM3 mRNA or CHRM3 protein expression, the kit comprising means for measuring the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one further gene associated therewith.

[0080] Preferably, the at least one further gene associated with CHRM3 mRNA expression is selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1.

[0081] A third embodiment of the present invention relates to the use of expression of CHRM3 mRNA or CHRM3 protein as a marker for the prognosis of a subject suffering from a disease or disorder, particularly colorectal cancer, based on the expression level of CHRM3 mRNA or CHRM3 protein by measuring the expression level of CHRM3 mRNA or CHRM3 protein and at least one gene associated with CHRM3 mRNA expression.

[0082] Preferably, the at least one further gene associated with CHRM3 mRNA expression is selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1.

[0083] A fourth embodiment of the present invention relates to a method for treating a subject suffering from a disease or disorder associated with overexpression of CHRM3 mRNA or CHRM3 protein, in particular colorectal cancer, wherein if the expression level of the CHRM3 mRNA or CHRM3 protein is significantly elevated compared to control levels, the subject is subjected to anti-tumor treatment.

[0084] There are a wide variety of anti-tumor therapies known to those skilled in the art. Based on certain factors, such as the nature of the cancer, the stage of the disease, etc., one skilled in the art can determine which therapy, e.g., surgery, radiation therapy, and / or chemotherapy, is appropriate for treating a subject in need of anti-tumor therapy.

[0085] Preferably, steps b) and c) further comprise measuring the expression level of at least one gene associated with CHRM3 mRNA or CHRM3 protein expression, preferably a gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1, and comparing the expression level of the gene with a control level.

[0086] The aforementioned rating scales (prognostic rating scales) can be used to determine whether a subject should be treated and / or monitored with an anti-tumor therapy.

[0087] Preferably, the expression levels of CHRM3 mRNA and at least one further associated gene are converted into a rating scale.

[0088] A fifth embodiment of the present invention relates to a method for treating a subject suffering from a disease or disorder associated with overexpression of CHRM3 mRNA or CHRM3 protein and activity of CHRM3 protein, particularly colorectal cancer. If the expression level of CHRM3 mRNA or CHRM3 protein and activity of CHRM3 protein are significantly elevated compared to control levels, the subject is treated with an effective amount of a drug that inhibits CHRM3 activity. The cancer-associated activity of CHRM3 is determined by measuring the expression of genes that exhibit CHRM3 activity. These genes are determined by inhibiting CHRM3 in cancer cells derived from the patient and determining which genes are significantly altered after inhibition. This is the therapeutic property of CHRM3.

[0089] Preferably, steps b) and c) further comprise measuring the expression level of at least one gene responsive to CHRM3 protein activity and comparing the expression level of the gene with a control level.

[0090] A rating scale (treatment rating scale) can be used to determine whether a particular subject should be treated with an agent that inhibits CHRM3 activity.

[0091] Preferably, the expression level of CHRM3 mRNA, the activity of CHRM3 protein and at least one further response gene are converted into a rating scale (therapeutic rating scale).

[0092] Preferably, the subject is further administered an effective amount of an EGFR inhibitor and / or an FGFR inhibitor.

[0093] Suitable EGFR inhibitors are preferably selected from erlotinib, gefitinib, osimertinib, cetuximab, and panitumumab.

[0094] Suitable FGFR inhibitors are preferably selected from erdafitinib, pemigatinib, and infigratinib.

[0095] As will be appreciated by those skilled in the art, the particular formulation will depend in part on the particular inhibitor or other chemotherapeutic agent used and the selected route of administration. Accordingly, there are many different suitable formulations of the composition. Suitable formulations for oral, parenteral, aerosol, transdermal, topical, or other modes of administration will be apparent to those skilled in the art.

[0096] Those skilled in the art can readily determine the appropriate dosage, schedule, and method of administration for the exact formulation of the composition used to achieve the desired anti-cancer effective amount or effective concentration of the drug in an individual patient. Those skilled in the art can also readily determine and use appropriate indicators of the "effective concentration" / "effective amount" of the inhibitor by direct or indirect analysis of appropriate patient samples (e.g., blood and / or tissue).

[0097] The dose of an inhibitor or composition thereof administered to an animal, particularly a human, should be sufficient to produce at least a therapeutic response in the individual within a reasonable time frame (an anticancer-effective dose). The precise amount administered will vary from subject to subject, depending on the subject's species, age, weight, and general condition, the severity or mechanism of the disease being treated, the specific agent or vehicle used, and its method of administration. The dosage used to achieve a desired anticancer concentration in the body will be determined by the potency of the specific inhibitor used, the pharmacodynamics associated with the agent in the host, the severity of the disease state of the infected individual, and, in the case of systemic administration, the weight and age of the individual. The size of the dose will also be determined by the presence of any adverse side effects that may accompany the specific inhibitor or composition thereof used. Minimizing adverse side effects whenever possible is generally desirable.

[0098] Preferably, the disease or disorder from which the subject is suffering is cancer, particularly cancer of epithelial cell origin, including primary cancer and secondary (metastatic) disease.

[0099] In particular, the cancer is selected from glioblastoma, melanoma, colon cancer, colorectal cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, bladder cancer, head cancer, neck cancer, and kidney cancer.

[0100] In particular, said disease or disorder is colorectal cancer.

[0101] Preferably, the CHRM3 mRNA inhibitor is selected from a small molecule inhibitor, a monoclonal antibody, and a combination thereof.

[0102] The small molecule inhibitor is preferably selected from darifenacin and zamifenacin. [Brief explanation of the drawings]

[0103] Figure 1: Elevated CHRM3 expression in colorectal cancer tumor samples compared to normal samples, as measured by RNA-Seq. Tumor samples (dark grey boxplot / right) show a statistically significant increase compared to normal samples.

[0104] Figure 2: Primary colon carcinoma samples from patients with high M3 receptor expression were grown as organoids in cell culture in the absence of the commonly added exogenous growth factors EGF and FGF. Organoid growth was inhibited by the M3-selective antagonist zamifenacin at concentrations consistent with the drug's M3 receptor affinity. The addition of the general M3 receptor agonist carbachol reversed the inhibition.

[0105] Figure 3: Kaplan-Meier curves based on progression-free survival and CHRM3 expression in patients with colorectal cancer tumors. Figure 3a: The tumor patients were divided into two cohorts based on their median CHRM3 expression. The lower line represents patients with CHRM3 expression above the median, and the upper line represents patients with CHRM3 expression below the median. As can be seen from this figure, patients with high expression have shorter survival times. Figure 3b: The tumor patients were divided into two cohorts based on their upper and lower quartiles of CHRM3 expression. The lower line represents patients with the top 25% of CHMR3 expression, and the upper line represents patients with the bottom 25% of CHRM3 expression. As can be seen from this figure, patients with high expression have shorter survival times.

Claims

1. 1. A method for predicting the prognosis of a subject suffering from colorectal cancer based on the expression level of CHRM3 mRNA, comprising: a) obtaining a sample from a tumor of a subject with colorectal cancer; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein with a control level. Including, A method in which a significant increase in the expression level of CHRM3 mRNA or protein compared to the control level indicates that the subject is likely to have a poor prognosis for the disease.

2. The method of claim 1, wherein steps b) and c) further comprise measuring the expression level of at least one gene associated with CHRM3 mRNA expression, preferably a gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1, and comparing the expression level of the at least one gene associated with CHRM3 mRNA expression with a control level.

3. The method according to claim 1 or 2, wherein the expression levels of the CHRM3 mRNA and optionally at least one further associated gene are converted into a (prognostic) evaluation scale.

4. 4. The method of claim 3, wherein the (prognostic) assessment scale is used to determine whether the subject should be treated with an anti-tumor therapy and / or monitored for progression of the disease.

5. A kit for predicting the prognosis of a subject suffering from colorectal cancer based on the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one further gene associated with CHRM3 mRNA expression, preferably a gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1, the kit comprising a means for measuring the expression level of CHRM3 mRNA or CHRM3 protein and optionally at least one gene associated therewith.

6. Use of expression of CHRM3 mRNA or CHRM3 protein as a marker for prognosis in a subject suffering from colorectal cancer, optionally comprising measuring the expression level of CHRM3 mRNA or CHRM3 protein using at least one additional gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1.

7. 1. A method for treating a subject suffering from colorectal cancer associated with overexpression of CHRM3 mRNA or CHRM3 protein, comprising: a) obtaining a sample from a tumor of a subject with colorectal cancer; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein with a control level. Including, If the expression level of the CHRM3 mRNA or CHRM3 protein is significantly elevated compared to the control level, the subject is subjected to anti-tumor treatment.

8. The method of claim 7, wherein steps b) and c) further comprise measuring the expression level of at least one gene associated with CHRM3 mRNA or CHRM3 protein expression, preferably a gene selected from LGR5, OLFM4, TRAF5, CD46, TGFB1, SLC5A1, and OXGR1, and comparing the expression level of the at least one gene associated with CHRM3 mRNA expression with a control level.

9. The method of claim 7 or 8, wherein the expression levels of CHRM3 mRNA or CHRM3 protein and optionally at least one further related gene are converted into a (prognostic) evaluation scale, and said (prognostic) evaluation scale is used to decide whether the subject should be treated with an anti-tumor therapy and / or monitored for the progression of the disease.

10. 1. A method for treating a subject suffering from colorectal cancer associated with overexpression of CHRM3 mRNA or CHRM3 protein and activity of CHRM3 protein, comprising: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the expression level of CHRM3 mRNA or CHRM3 protein and the activity of CHRM3 protein; c) comparing the expression level of the CHRM3 mRNA or CHRM3 protein and the activity of the CHRM3 protein with control levels. Including, If the expression level of CHRM3 mRNA or CHRM3 protein and the activity of CHRM3 protein are significantly increased compared to the control levels, the subject is treated with an effective amount of a drug that inhibits CHRM3 activity.

11. The method of claim 10, wherein steps b) and c) further comprise measuring the expression level of at least one response gene to an effective amount of CHRM3 protein activity and comparing the expression level of the at least one response gene with a control level.

12. The method according to claims 10 to 11, wherein the level of CHRM3 protein activity and optionally at least one further response gene is converted into a (therapeutic) assessment scale, and said (therapeutic) assessment scale is used to determine whether the subject should be treated with an effective amount of a drug that inhibits CHRM3 activity.

13. The method according to claims 10 to 12, wherein an effective amount of an EGFR inhibitor and / or an FGFR inhibitor is further administered.

14. 10. The method of any one of the preceding claims, wherein the colorectal cancer includes primary and secondary metastatic disease.

15. 10. The method of claim 1, wherein the subject is a human.

16. The method of claims 10 to 15, wherein the CHRM3 mRNA inhibitor is selected from a small molecule inhibitor, a monoclonal antibody, or a combination thereof.

17. The method of claims 10 to 16, wherein the subject is a human patient suffering from colorectal cancer and the CHRM3 mRNA inhibitor is selected from darifenacin or zamifenacin and mixtures thereof.

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