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Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Although overall survival for CRC patients has improved with earlier screening, survival continues to vary substantially across stages. Also, while the introduction of targeted therapies, including VEGF and EGFR inhibitors, has contributed to improving survival, better tools are needed to optimize patient selection and maximize therapeutic benefits. Emerging biomarkers can be used to guide pharmacologic decision-making, as well as monitor treatment response, clarify the need for adjuvant therapies, and indicate early signs of recurrence. This is a narrative review examining the current and evolving use of predictive and prognostic biomarkers in colorectal cancer.Areas covered include mutations of the MAPK (KRAS, BRAF) and HER2 pathways and their impacts on treatment decisions. In addition, novel methods for assessing tumor mutations and tracking treatment responses are examined.The standard of care pathway for staging, and treatment selection and surveillance, of CRC will expand to include novel biomarkers in the next 5 years. It is anticipated that these new biomarkers will assist in decision-making regarding selection of targeted therapies and, importantly, in risk stratification for treatment decisions in patients at high risk for recurrence.

The mammalian genome encodes seven guanylyl cyclases, GC-A to GC-G, that are homodimeric transmembrane receptors activated by a diverse range of endogenous ligands. These enzymes convert guanosine-5'-triphosphate to the intracellular second messenger cyclic guanosine-3',5'-monophosphate (cyclic GMP). GC-A, GC-B and GC-C are expressed predominantly in the cardiovascular system, skeletal system and intestinal epithelium, respectively. GC-D and GC-G are found in the olfactory neuropepithelium and Grueneberg ganglion of rodents, respectively. GC-E and GC-F are expressed in retinal photoreceptors.

Figure S11 shows that hGUCY2C-specific 5F9.m28BBz CAR-T cells do not recognize murine GUCY2C.

Cyclic GMP responses in transverse colon biopsies in subjects receiving linaclotide (Active) or placebo.

Incidence of adverse events by study age.

Cyclic GMP responses in rectal biopsies in subjects receiving linaclotide (Active) or placebo.

Pre-Intervention Colonoscopy/Endoscopy Results.

Post intervention colonoscopy/endoscopy results.

Cyclic GMP responses in cecal biopsies in subjects receiving linaclotide (Active) or placebo.

Baseline Physical Exam.

Chronic idiopathic constipation and irritable bowel syndrome with constipation are complex, overlapping conditions. Although multiple guidelines have informed healthcare providers on appropriate treatment options for patients with chronic idiopathic constipation and irritable bowel syndrome with constipation, little direction is offered on treatment selection. First-line treatment options usually include fiber and over-the-counter osmotic laxatives; however, these are insufficient for many individuals. When these options fail, prescription secretagogues (plecanatide, linaclotide, lubiprostone, and tenapanor [pending commercial availability]), or serotonergic agents (prucalopride and tegaserod) are generally preferred. Individuals experiencing concurrent abdominal pain and/or bloating may experience greater overall improvements from prescription therapies because these agents have been proven to reduce concurrent abdominal and bowel symptoms. Should initial prescription treatments fail, retrying past treatment options (if not adequately trialed initially), combining agents from alternative classes, or use of adjunctive therapies may be considered. Given the broad spectrum of available agents, therapy should be tailored by mutual decision-making between the patient and practitioner. Overall, patients need to be actively monitored and managed to maximize clinical outcomes.

High doses of ionizing radiation induce acute damage to epithelial cells of the gastrointestinal (GI) tract, mediating toxicities restricting the therapeutic efficacy of radiation in cancer and morbidity and mortality in nuclear disasters. No approved prophylaxis or therapy exists for these toxicities, in part reflecting an incomplete understanding of mechanisms contributing to the acute radiation-induced GI syndrome (RIGS). Guanylate cyclase C (GUCY2C) and its hormones guanylin and uroguanylin have recently emerged as one paracrine axis defending intestinal mucosal integrity against mutational, chemical, and inflammatory injury. Here, we reveal a role for the GUCY2C paracrine axis in compensatory mechanisms opposing RIGS. Eliminating GUCY2C signaling exacerbated RIGS, amplifying radiation-induced mortality, weight loss, mucosal bleeding, debilitation, and intestinal dysfunction. Durable expression of GUCY2C, guanylin, and uroguanylin mRNA and protein by intestinal epithelial cells was preserved following lethal irradiation inducing RIGS. Oral delivery of the heat-stable enterotoxin (ST), an exogenous GUCY2C ligand, opposed RIGS, a process requiring p53 activation mediated by dissociation from MDM2. In turn, p53 activation prevented cell death by selectively limiting mitotic catastrophe, but not apoptosis. These studies reveal a role for the GUCY2C paracrine hormone axis as a novel compensatory mechanism opposing RIGS, and they highlight the potential of oral GUCY2C agonists (Linzess; Trulance) to prevent and treat RIGS in cancer therapy and nuclear disasters. Cancer Res; 77(18); 5095–106. ©2017 AACR.

Purpose: Lymph node involvement by histopathology informs colorectal cancer prognosis, whereas recurrence in 25% of node-negative patients suggests the presence of occult metastasis. GUCY2C (guanylyl cyclase C) is a marker of colorectal cancer cells that identifies occult nodal metastases associated with recurrence risk. Here, we defined the association of occult tumor burden, quantified by GUCY2C reverse transcriptase-PCR (RT-PCR), with outcomes in colorectal cancer.Experimental Design: Lymph nodes (range: 2–159) from 291 prospectively enrolled node-negative colorectal cancer patients were analyzed by histopathology and GUCY2C quantitative RT-PCR. Participants were followed for a median of 24 months (range: 2–63). Time to recurrence and disease-free survival served as primary and secondary outcomes, respectively. Association of outcomes with prognostic markers, including molecular tumor burden, was estimated by recursive partitioning and Cox models.Results: In this cohort, 176 (60%) patients exhibited low tumor burden (MolLow), and all but four remained free of disease [recurrence rate 2.3% (95% CI, 0.1–4.5%)]. Also, 90 (31%) patients exhibited intermediate tumor burden (MolInt) and 30 [33.3% (23.7–44.1)] developed recurrent disease. Furthermore, 25 (9%) patients exhibited high tumor burden (MolHigh) and 17 [68.0% (46.5–85.1)] developed recurrent disease (P < 0.001). Occult tumor burden was an independent marker of prognosis. MolInt and MolHigh patients exhibited a graded risk of earlier time to recurrence [MolInt, adjusted HR 25.52 (11.08–143.18); P < 0.001; MolHigh, 65.38 (39.01–676.94); P < 0.001] and reduced disease-free survival [MolInt, 9.77 (6.26–87.26); P < 0.001; MolHigh, 22.97 (21.59–316.16); P < 0.001].Conclusion: Molecular tumor burden in lymph nodes is independently associated with time to recurrence and disease-free survival in patients with node-negative colorectal cancer. Clin Cancer Res; 17(10); 3293–303. ©2011 AACR.

Supplementary Figure 1 Legend

Supplementary Figure 1. Silencing p53 expression in intestine of p53int-/- mice eliminates phospho-p53 responses to DNA damage induced by radiation. Supplementary Figure 2. Silencing p53 expression in intestine of p53int-/- mice amplifies RIGS-related toxicity. Supplementary Figure 3. Littermates are utilized to minimize the effect of genetic backgrounds in transgenic mice. Supplementary Figure 4. HCT116 cells do not exhibit GUCY2C ligand binding. Supplementary Figure 5. Total body irradiation induces gastrointestinal injury. Supplementary Figure 6. Hormone expression is preserved in RIGS in Gucy2c-/- mice. Supplementary Figure 7. Oral ST fails to oppose RIGS in the absence of GUCY2C. Supplementary Figure 8. Silencing p53 expression in intestine of p53int-/- mice does not affect apoptosis in RIGS.

Supplementary Figure 1 - Time to recurrence in stage I (blue), II (yellow), and III (black) patients with colorectal cancer.

CCR Translation for This Article from Occult Tumor Burden Predicts Disease Recurrence in Lymph Node–Negative Colorectal Cancer

Supplementary Figure Legends

Supplementary Figures S1-S3; Supplementary Materials and Methods.

Background: Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer.Methods: Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium.Results: Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100- to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient = 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors.Conclusions: Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells.Impact: Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy. Cancer Epidemiol Biomarkers Prev; 23(11); 2328–37. ©2014 AACR.

Body weights and guanylin expression in C57BL/6 mice on a lean or high fat (HF) diet, or switched from a high fat to a lean diet (HF-Lean).

Mouse diet calorie and nutrient content.

Legend for Supplementary Figures S1-S7.

INTRODUCTION: Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. There have been improvements in screening, and therefore overall survival, but patients continue to present at late stages when minimal treatment options are available to them. While some targeted therapies have been introduced, their application is limited by patient-specific tumor characteristics. Additional targets for CRC in patients who present at a late stage, or who experience tumor relapse, need to be identified to continue to improve patient outcomes. AREAS COVERED: This review focuses on emerging pathways and drug targets for the treatment of colorectal cancer. The shift to the cancer stem cell model and potential targets involving Wnt, NF-κB, phosphodiesterases, RAS, and guanylyl cyclase C, are discussed. The current utility of checkpoint inhibitors and evolving immunological options are examined. EXPERT OPINION: Surgery and current systemic cytotoxic therapies are inadequate to appropriately treat the full spectrum of CRC, especially in those patients who present with metastatic or treatment-refractory disease. In addition to the identification of new, more generalizable targets, additional focus is being placed on novel administrations. Immuno-oncologic options and stem cell-targeting therapies for mCRC will become available to patients and may increase survival.

Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we reveal that intestinal GUCY2C is selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2CHigh neuropod cells associate with co-cultured dorsal root ganglia neurons and induce hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient, but not GUCY2C-deficient, neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling and VP that was comparable to chemically-induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.

Colorectal cancer remains a major cause of mortality in the USA, despite advances in prevention and screening. Existing therapies focus primarily on generic treatment such as surgical intervention and chemotherapy, depending on disease severity. As personalized medicine and targeted molecular oncology continue to develop as promising treatment avenues, there has emerged a need for effective targets and biomarkers of colorectal cancer. The transmembrane receptor guanylyl cyclase C (GUCY2C) regulates intestinal homeostasis and has emerged as a tumor suppressor. Further, it is universally expressed in advanced metastatic colorectal tumors, as well as other cancer types that arise through intestinal metaplasia. In this context, GUCY2C satisfies many characteristics of a compelling target and biomarker for gastrointestinal malignancies.Colorectal cancer is a leading cause of death in the USA. In recent years, there has been a shift in the field of oncology from generic treatments, such as surgery and chemotherapy, to personalized molecular therapies, which focus on targeting specific attributes of each patient's unique cancer. Guanylyl cyclase C is a receptor expressed in the intestinal tract, where it regulates fluid secretion and prevents tumor formation. Beyond its function in the healthy intestine, it is expressed in colorectal tumors, and other types of cancer, where it regulates transformation. Therefore, guanylyl cyclase C can serve as a useful target in cancer for prevention and therapy, as well as a marker for tumor cell detection.

INTRODUCTION: Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and is the second leading cause of cancer-related death in the United States. Despite advances in early detection, ~25% of patients are late stage, and treated patients have

INTRODUCTION: Gastrointestinal (GI) cancers account for the second leading cause of cancer-related deaths in the United States. Guanylyl cyclase C (GUCY2C) is an intestinal signaling system that regulates intestinal fluid and electrolyte secretion as well as intestinal homeostasis. In recent years, it has emerged as a promising target for chemoprevention and therapy for GI malignancies. AREAS COVERED: The loss of GUCY2C signaling early in colorectal tumorigenesis suggests it could have a significant impact on tumor initiation. Recent studies highlight the importance of GUCY2C signaling in preventing colorectal tumorigenesis using agents such as linaclotide, plecanatide and sildenafil. Further, GUCY2C is a novel target for immunotherapy and a diagnostic marker of primary and metastatic disease. EXPERT OPINION: There is an unmet need for prevention and therapy in GI cancers. In that context, GUCY2C is a promising target for prevention, although the precise mechanisms by which GUCY2C signaling affects tumorigenesis remain to be defined. Further, clinical trials are exploring its role as an immunotherapeutic target for vaccines to prevent metastatic disease. Indeed, GUCY2C is an emerging target across the disease continuum from chemoprevention, to diagnostic management, through the treatment and prevention of metastatic disease.

Summary Background Chronic constipation is a common, heterogeneous disorder with multiple symptoms and pathophysiological mechanisms. Patients are often referred to a gastroenterology provider after laxatives fail. However, there is limited knowledge of the spectrum and management of constipation disorders. Aim To discuss the latest understanding of the spectrum of constipation disorders, tools for identifying a pathophysiologic‐based diagnosis in the specialist setting, treatment options and the patient's perspective of constipation. Methods Literature searches were conducted using PubMed for constipation diagnostic criteria, diagnostic tools and approved treatments. The authors provided insight from their own practices. Results Clinical assessment, stool diaries and Rome IV diagnostic criteria can facilitate diagnosis, evaluate severity and distinguish between IBS with constipation, chronic idiopathic constipation and dyssynergic defecation. Novel smartphone applications can help track constipation symptoms. Rectal examinations, anorectal manometry and balloon expulsion, assessments of neuromuscular function with colonic transit time and colonic manometry can provide mechanistic understanding of underlying pathophysiology. Treatments include lifestyle and diet changes, biofeedback therapy and pharmacological agents. Several classes of laxatives, as well as prokinetic and prosecretory agents, are available; here we describe their mechanisms of action, efficacy and side effects. Conclusions Constipation includes multiple overlapping subtypes identifiable using detailed history, current diagnostic tools and smartphone applications. Recognition of individual subtype(s) could pave the way for optimal, evidence‐based treatments by a gastroenterology provider., Gastroenterology providers should be familiar with the constipation subtypes and their underlying pathophysiology, as well as key signalling pathways that may contribute to constipation. Familiarisation with the varied mechanisms of action, expected efficacy and side effects of available therapies helps to select the appropriate treatment and meet patient expectations.

The mammalian genome encodes seven guanylyl cyclases, GC-A to GC-G, that are homodimeric transmembrane receptors activated by a diverse range of endogenous ligands. These enzymes convert guanosine-5'-triphosphate to the intracellular second messenger cyclic guanosine-3',5'-monophosphate (cyclic GMP). GC-A, GC-B and GC-C are expressed predominantly in the cardiovascular system, skeletal system and intestinal epithelium, respectively. GC-D and GC-G are found in the olfactory neuropepithelium and Grueneberg ganglion of rodents, respectively. GC-E and GC-F are expressed in retinal photoreceptors.

Most sporadic colorectal cancer reflects acquired mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, while germline heterozygosity for mutant APC produces the autosomal dominant disorder Familial Adenomatous Polyposis (FAP) with a predisposition to colorectal cancer. In these syndromes, loss of heterozygosity (LOH) silences the remaining normal allele of APC, through an unknown mechanism, as the initiating step in transformation. Guanylyl cyclase C receptor (GUCY2C) and its hormones, uroguanylin and guanylin, have emerged as a key signaling axis opposing mutations driving intestinal tumorigenesis. Indeed, uroguanylin and guanylin are among the most commonly repressed genes in colorectal cancer. Here, we explored the role of APC heterozygosity in mechanisms repressing hormone expression which could contribute to LOH. In genetic mouse models of APC loss, uroguanylin and guanylin expression were quantified following monoallelic or biallelic deletion of the Apc gene. Induced biallelic loss of APC repressed uroguanylin and guanylin expression. However, monoallelic APC loss in Apcmin/+ mice did not alter hormone expression. Similarly, in FAP patients, normal colonic mucosa (monoallelic APC loss) expressed guanylin while adenomas and an invasive carcinoma (biallelic APC loss) were devoid of hormone expression. Thus, uroguanylin and guanylin expression by normal intestinal epithelial cells persists in the context of APC heterozygosity and is lost only after tumor initiation by APC LOH. These observations reveal a role for loss of the hormones silencing the GUCY2C axis in tumor progression following biallelic APC loss, but not in mechanisms creating the genetic vulnerability in epithelial cells underlying APC LOH initiating tumorigenesis.

Sporadic colorectal cancer initiates with mutations in APC or its degradation target β-catenin, producing TCF-dependent nuclear transcription driving tumorigenesis. The intestinal epithelial receptor, GUCY2C, with its canonical paracrine hormone guanylin, regulates homeostatic signaling along the crypt-surface axis opposing tumorigenesis. Here, we reveal that expression of the guanylin hormone, but not the GUCY2C receptor, is lost at the earliest stages of transformation in APC-dependent tumors in humans and mice. Hormone loss, which silences GUCY2C signaling, reflects transcriptional repression mediated by mutant APC-β-catenin-TCF programs in the nucleus. These studies support a pathophysiological model of intestinal tumorigenesis in which mutant APC-β-catenin-TCF transcriptional regulation eliminates guanylin expression at tumor initiation, silencing GUCY2C signaling which, in turn, dysregulates intestinal homeostatic mechanisms contributing to tumor progression. They expand the mechanistic paradigm for colorectal cancer from a disease of irreversible mutations in APC and β-catenin to one of guanylin hormone loss whose replacement, and reconstitution of GUCY2C signaling, could prevent tumorigenesis