Guanylyl Cyclase 2C (GUCY2C) in Gastrointestinal Cancers: Recent Innovations and Therapeutic Potential

Abstract

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.

1. Introduction

Guanylyl cyclase C (GUCY2C), is a transmembrane receptor predominantly expressed apically on intestinal crypt and villus cells [1]. GUCY2C was originally identified in the 1970s as the receptor for ST, a heat-stable enterotoxin produced by diarrheagenic enteric bacteria such as enterotoxigenic Escherichia coli (ETEC) [2,3]. Since its discovery, the loss or gain of function of GUCY2C has been implicated in several disorders associated with constipation or diarrhea [4–7]. Moreover, GUCY2C signaling has been targeted as a therapy to treat gastrointestinal (GI) disorders common worldwide, including irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC), as well as inflammatory bowel disease (IBD) [8,9].

GUCY2C is activated by endogenous hormone ligands uroguanylin, primarily expressed in the small intestine, and guanylin, primarily expressed in the large intestine [10,11]. Both hormones activate GUCY2C to initiate a cascade of downstream signaling that regulates fluid and electrolyte homeostasis, maintenance of intestinal barrier integrity, and intestinal tumorigenesis [10–14].

Together, GI cancers, including cancers of the colon and rectum (colorectal), esophagus and stomach (gastroesophageal), liver, gallbladder, pancreas, small intestine, appendix, and anus, represent the greatest number of new cases of cancer in the United States annually [15]. Alone, colorectal cancer is the fourth most commonly diagnosed cancer in the United States [15]. Alarmingly, trends in colorectal cancer suggest that the number of patients under the age of 50 diagnosed with advanced-stage disease has been increasing over the past 25 years, highlighting the critical need for improved therapies [16]. Current treatment strategies depend on disease stage. The primary treatment for localized disease (stage I-II) is complete surgical resection, while for stage III disease, adjuvant chemotherapy, commonly an oxaliplatin-based regimen, is administered [17]. Still, 5-year relative survival rate for colorectal cancer patients is 66%. Moreover, other GI cancer patients have lower outcomes. The 5-year relative survival rate for gastric, esophageal, and pancreatic cancers are 32%, 21% and 10%, respectively [15]. Despite novel advances in treatment, GI cancers together are still the second-leading cause of cancer-associated death in the United States, highlighting the need for effective prevention and therapy [15].

The hormone ligands for the receptor GUCY2C are among the earliest lost gene products in tumorigenesis [18]. Therefore, much time and attention has been focused on understanding the relationship between GUCY2C signaling and colorectal cancer susceptibility. Here, we will review the signaling pathways in the healthy GI tract, including GUCY2C, and the use of GUCY2C signaling as a target to prevent and treat GI cancers.

2. Healthy gastrointestinal tract

Measuring about five meters in length, the GI tract is a vital barrier against infections and toxins and provides an environment for nutrient and water absorption necessary for survival [19]. Food travels from the oral cavity through the GI tract to be absorbed, digested, and excreted. The esophagus is a muscular tube connecting the oral cavity to the stomach for food and fluid transportation. The stomach mixes ingested food with water and gastric enzymes to initiate the process of digestion. The small intestine continues the process of digestion, mixing stomach contents with pancreatic enzymes in the duodenum, and absorbing the resulting elementary nutrients in the jejunum and ileum [20]. While the apical surface of the single layer of epithelial cells in the intestine is a source of nutrient absorption and fluid secretion in the lumen, the basolateral surface sits on the underlying lamina propria, a loose connective tissue that provides a source of immune defense and vascular support to the epithelium [21]. Absorption in the epithelium is maximized by a large epithelial surface area achieved by invaginations in the mucosa, known as crypts, and projections into the small intestinal lumen, known as villi [20,22]. This epithelium is one of the few in the body that continuously regenerates. At the base of the crypt, there are long-lived stem cells that develop into proliferating daughter cells in the transit amplifying zone that differentiate into specialized epithelial cell subtypes and migrate upwards from the crypt to the tip of the villus [22]. Specialized epithelial cell subtypes include nutrient-absorbing enterocytes, mucus-secreting goblet cells, and hormone-secreting enteroendocrine cells. Additionally, Paneth cells migrate downwards into the crypt, where they act to nourish stem cells and secrete antimicrobial substances into the lumen (Figure 1) [23]. Differentiated epithelial cells complete the cycle of regeneration, differentiation, and migration, in the span of 3–5 days, after which they slough off into the intestinal lumen [22]. Contrasting with the small intestine, the colon lacks villi, consistent with its role in fluid absorption and stool transit rather than nutrient absorption [22]. However, the large intestine contains crypts similar to those in the small intestine, with stem cells at the base that differentiate and migrate upwards to the crypt surface [22].

Figure 1.

The intestinal crypt-villus axis. The intestinal epithelium is composed of stem cells, transit amplifying (TA) cells, enterocytes, goblet cells, enteroendocrine cells, enteroendocrine cells with neuropods, and Paneth cells. The small intestine is organized into villus projections into the lumen and crypt invaginations into the lamina propria, while the colon is organized exclusively into crypts. Lgr5+ intestinal stem cells (ISCs) reside at the bottom of the crypt (position +1) and are supported by downward migrating Paneth cells. ISCs from the base of the crypt migrate upwards and give rise to quiescent, position +4 ISCs, or give rise to daughter cells in the TA zone that differentiate into specialized epithelial subtypes. As cells migrate upwards, they move from a Wnt-enriched to a BMP/Hedgehogenriched environment. Uroguanylin is enriched in the villus of the small intestine and guanylin is enriched in the upper portion of the crypt in the colon. BMP, bone morphogenetic protein; ISC, intestinal stem cell; TA, transit amplifying. Created with BioRender.com

Due to the rapid cell turnover in both small and large intestines, proliferation is a highly regulated feature. There are a number of factors regulating proliferation, including APC/β-catenin, epidermal growth factor (EGF), and bone morphogenic protein (BMP) signaling [24]. APC/β-catenin signaling is regulated by the extracellular ligand Wnt. Wnt hormones are secreted at the base of the crypt, providing an environment favorable for stem cell renewal and epithelial proliferation (Figure 1). As stem cells migrate upwards in the crypt, they leave the stem cell-forward environment and Wnt-driven effects are reduced. Conversely, Hedgehog and BMP effects increase along their concentration gradients, supporting differentiation into the various specialized epithelial cells of the small and large intestines and, ultimately, senescence (Figure 1) [25].

In the APC/β-catenin signaling pathway, cytosolic β-catenin enters a multi-protein complex stabilized by the scaffold proteins adenomatous polyposis coli (APC) and axin. The serine/threonine kinases casein kinase 1a and glycogen synthase kinase 3 phosphorylate β-catenin, marking it for poly-ubiquitination by the β-TrCP E3 ubiquitin ligase and subsequent degradation by the proteasome. In the presence of extracellular Wnt, this process is blocked. Wnt binds to its cell surface receptor, Frizzled, and co-receptor, LRP5/6. Axin is recruited to the plasma membrane, preventing the destruction complex from forming. Subsequently, cytosolic β-catenin accumulates and translocates to the nucleus, where it associates with the T-cell factor (TCF) family of nuclear transcription factors, activating an oncogenic transcriptional program [26].

3. GUCY2C signaling in the healthy intestine

Guanylyl cyclases are classified by localization in either the particulate (membrane-bound) or soluble (cytosolic) fractions of the cell. Peptide ligands, Ca2+ transients, and nitric oxide activate guanylyl cyclases [27]. Guanylyl cyclase C, GUCY2C, is a transmembrane receptor predominantly expressed apically on intestinal crypt and villus cells [27]. The endogenous hormone ligands uroguanylin and guanylin are synthesized as propeptides by secretory epithelial cells and processed into biologically active 16-mer (uroguanylin), or 15-mer (guanylin), peptides [28–31]. Structurally similar, uroguanylin and guanylin each have two disulfide bridges critical for stability and resistance to denaturation in the intestine [10,32–34].

GUCY2C is activated by ligand binding to its extracellular domain, inducing the catalytic domain to convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). Elevated intracellular cGMP drives phosphorylation and translocation of the cystic fibrosis transmembrane conductance regulator (CFTR) to the cell surface, prompting Cl⁻ and HCO₃⁻ efflux into the intestinal lumen. Further, cGMP signaling inhibits the apical Na⁺/H⁺ exchanger 3 (NHE3), preventing Na⁺ absorption from the lumen. The combined electrolyte efflux and retention in the lumen initiates an osmotic gradient that drives intestinal fluid secretion [35].

Cyclic GMP alters cellular functions through three cGMP-binding proteins: cGMP-modulated cation channels, cGMP-regulated phosphodiesterases (PDEs), and cGMP-dependent protein kinases (cGKs) [36]. Cyclic nucleotide-gated ion channels contain cyclic nucleotide binding domains (CNBs). One such channel is NHE3, which is regulated by direct cyclic nucleotide binding, in addition to phosphorylation by cAMP- and cGMP-dependent protein kinases. Cyclic GMP-mediated inhibition reduces the activity of the NHE exchanger that prevents sodium and chloride ion and water absorption from the intestinal lumen [36].

Additionally, PDEs regulate intracellular cAMP and cGMP levels through their cyclic nucleotide hydrolase activities [37]. Of the 11 identified PDEs, many are expressed in the intestine and catalyze the hydrolysis of cAMP and/or cGMP. For example, PDE2A, PDE3A/3B, PDE10A and PDE11A hydrolyze both cGMP and cAMP. PDE4 and PDE8 are cAMP-specific PDEs, while PDE5 and PDE9 are specific for cGMP [38]. Collectively, PDEs act as critical regulators of cGMP-mediated physiological responses [36]. For example, cGMP binds to and activates the regulatory domain of PDE5, providing a negative feedback loop since PDE5 degrades cGMP (Figure 2). Additionally, PDEs can be a source of cross-regulation between cAMP and cGMP. PDE3 activity is inhibited by cGMP, which elevates intracellular cAMP concentrations and activates cAMP-dependent protein kinase (PKA) signaling (Figure 2) [38].

Figure 2.

GUCY2C signaling in the healthy intestinal cell. GUCY2C is activated by its endogenous or exogenous ligands, stimulating conversion of GTP into cGMP. The cGMP effector, PKGII, promotes anion secretion by the CFTR transporter and inhibits sodium ion absorption by the NHE3 transporter, thereby producing an ion and fluid gradient into the intestinal lumen. Additionally, cGMP directly inhibits NHE3. Cyclic GMP inhibits PDE3 and cross-activates cAMP/PKA signaling to further promote anion secretion by the CFTR transporter. Cyclic GMP also activates PDE5, promoting the degradation of cGMP. cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; cGMP, cyclic guanosine monophosphate; GTP, guanosine triphosphate; GUCY2C, guanylyl cyclase C; NHE3, Na+/H+ exchanger III; PDE3, phosphodiesterase 3; PDE5, phosphodiesterase 5, PKA, protein kinase A; PKGII, protein kinase G II; ST, bacterial heat-stable enterotoxin; 5’- AMP, 5’-adenosine monophosphate; 5’-GMP, 5’-guanosine monophosphate. Created with BioRender.com

Finally, cGMP regulates cGMP-dependent protein kinase (PKG), a serine/threonine kinase, which is the main downstream effector molecule for cGMP (Figure 2) [39]. There are two isoforms, PKGI and PKGII, which are expressed throughout the body, including in the intestine. PKGI is expressed in smooth muscle cells, where it regulates intestinal contractility, while PKGII is predominantly expressed in the intestinal epithelium [40]. PKG is active as a homodimer and has a regulatory N‐terminus domain that inhibits the activity of the kinase domain. Cyclic GMP binding to the regulatory N-terminus domain induces an activating conformational change. In the intestine, PKGII phosphorylates CFTR and cross-activates protein kinase A to stimulate the efflux of Cl^- and HCO₃^- ions into the gut lumen and promoting fluid secretion. Additionally, PKGII phosphorylates NHE3 to inhibit sodium absorption and prevent fluid absorption (Figure 2) [41].

Although there is evidence that cGMP plays a role in tumor suppression, the mechanism by which this occurs remains unclear. Cyclic GMP‐dependent protein kinases are likely to play an important role, as expression of both PKGI and PKGII are reduced in colorectal carcinomas [42]. Furthermore, inhibitors of cGMP-specific PDE5 elevate cGMP levels in colorectal cancer cells, inducing apoptosis and preventing tumor growth, which will be discussed below.

4. Dysregulated pathways in GI cancers

GI cancers arise from a combination of genetic and environmental risk factors. Inactivating mutations of APC are associated with ~80% of all colon tumors [26], and are found in other GI cancer types such as gastric cancer [43]. In colorectal cancers of this type, loss of function in both APC alleles is a necessary step for tumor initiation. APC is unable to regulate β-catenin protein stability, which subsequently causes uncontrolled β-catenin nuclear signaling, driving tumorigenic gene expression [26]. Truncating mutations in APC remove all binding sites for axin, which binds β-catenin and recruits the protein kinases GSK3 and CKI to label β-catenin for degradation by β-TrCP E3 ubiquitin ligase. Loss of regulation of β-catenin also occurs in cases where β-catenin is mutated in exon 3, preventing it from being marked for degradation by GSK3 and CKI [26]. Although APC/β-catenin signaling is an attractive target for GI cancers, these molecules have proven difficult to impact with pharmacotherapy.

Greater than 50% of cases of colorectal cancer can be attributed to modifiable lifestyle and environmental risk factors such as cigarette smoking, physical inactivity, heavy alcohol consumption, obesity, and poor diet [44]. These environmental influences can impact the gut microbiota, which consists of about 10 trillion microbial cells including bacteria, fungi, protozoa, and viruses[45]. In healthy intestine, microbes support digestion and metabolism, pathogen barrier, inflammation, and immunity[46]. Recently, the relationship between gut dysbiosis and intestinal malignancy has been interrogated. In CRC patients, an enrichment of bacteria such as Fusobacterium nucleatum, Streptococcus bovis, and enterotoxigenic Bacteroides fragilis, has been linked to colorectal pathogenesis through increased cell proliferation, inflammation, and DNA damage [46]. Further, a causal relationship between gut dysbiosis and response to immune therapy has been identified. Recent studies have linked failure to respond to anti-PD-1 therapy in patients to abnormal gut microbiota [47,48]. Additionally, Fusobacterium nucleatum has been associated with resistance to chemotherapy agent oxaliplatin via induction of autophagy through Toll-like receptor 4 [49]. Therefore, there is evidence that analysis of gut microbiome can drive advances to personalize and enhance immunotherapy.

Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) that are associated with risk of colorectal cancer. Gene-environment (GxE) interaction between these genetic variants and environmental risk factors may modify cancer onset and progression [50]. Indeed, an interaction associated with an increased risk of colorectal cancer was identified between GWAS locus, rs4143094 (10p14), and processed meat consumption [51]. GxE interactions are critical in the emerging field of molecular pathological epidemiology (MPE), the interdisciplinary study of pathology and data science. MPE utilizes a big data approach to explore interactions between the environment, tumor, and host for prevention, identification, and treatment of cancers [52,53]. The field subdivides into areas such as pharmaco-MPE, immune-MPE, and microbial-MPE to subtype tumors for more accurate treatment of cancers and predictions of clinical outcomes. Further MPE studies should be utilized to bridge current research gaps in precision medicine.

5. Loss of GUCY2C signaling in colorectal cancer

GUCY2C signaling was first associated with colorectal cancer through population studies suggesting an inverse relationship between colorectal cancer prevalence and ETEC infections [54]. ETEC infections produce heat-stable enterotoxins, STs, that activate GUCY2C signaling to cause Traveler’s diarrhea [2,3,55]. Further, GUCY2C signaling has been implicated in colorectal cancer through the loss of the receptor’s endogenous ligands. In a study of nearly 300 tumors and matched normal adjacent tissues, guanylin mRNA was lost in more than 85% of tumors compared with matched normal epithelia [18]. Moreover, the ligands for GUCY2C are among the earliest gene products lost in colorectal cancer [18,56]. Interestingly, it was observed recently that loss of guanylin in mice is a direct downstream event of mutant APC/β-catenin signaling [57]. Further, loss of heterozygosity (biallelic loss) of APC is necessary for loss of guanylin hormone expression [58]. Together, these data suggest that GUCY2C signaling may play a role in early colorectal tumorigenesis in direct association with mutant APC/β-catenin signaling.

Studies utilizing mice deficient in GUCY2C (Gucy2c^−/−) have been instrumental in elucidating the role of that receptor in colorectal cancer [59]. Loss of GUCY2C is associated with a tumorigenic phenotype of epithelial dysfunction in which there is increased cellular proliferation and migration, DNA damage, and metabolic reprogramming [12–14,60]. Loss of GUCY2C, or its hormone ligand guanylin, increases proliferation resulting in greater crypt length, PCNA⁺ staining, and epithelial cell migration rate [61,62]. Further, mice deficient in GUCY2C have increased oxidative DNA damage and apoptotic cells compared to mice with normal GUCY2C expression [12,14]. Finally, eliminating GUCY2C expression produces metabolic reprogramming in Gucy2c^−/− mice. Indeed, mitochondrial quantity and function are reduced in intestinal cells of mice deficient in GUCY2C. Further, enzymes mediating glycolysis in tumors, such as hexokinase II and glucose transporter I, are upregulated in Gucy2c^−/− mice [14].

Conversely, GUCY2C activating ligands suppress oncogenic drivers such as cyclin D1, β-catenin, and pAKT, and activate tumor suppressor genes such as p21 and p27 [13,14]. Indeed, activation of GUCY2C by the bacterial enterotoxin ST in a human colon cancer line resulted in accumulation of p21 in the nucleus. The cyclin-dependent kinase inhibitor p27 is decreased in mice deficient in GUCY2C, while activation of GUCY2C by ST increased p27 and reduced cyclin D1 and phosphorylated retinoblastoma (pRb) expression. Further, mice deficient in GUCY2C exhibit activated AKT, often observed in the context of colorectal tumorigenesis. Together, these data suggest that loss of GUCY2C signaling could contribute to the onset of colorectal cancer and may be targetable for colorectal cancer prevention and treatment.

6. Ligand replacement therapy

The loss of guanylin hormone early in colorectal cancer, together with the intestinal epithelial dysfunction that occurs in the GUCY2C-deficient mouse model, suggests that colorectal cancer may arise within an environment of ligand deficiency that silences GUCY2C signaling [18,61]. This suggests that reconstituting GUCY2C signaling with exogenous ligands might be used as a strategy to regulate intestinal cell proliferation to prevent tumorigenesis. In that context, diet-induced obesity suppresses guanylin expression supporting tumorigenesis in mice, and that transformation can be blocked by overexpression of guanylin [63]. Additionally, in an Apc^min/+ mouse model, oral uroguanylin administration inhibits tumorigenesis, suggesting that activation of GUCY2C signaling might be a strategy for cancer chemoprevention [64].

Food and Drug Administration (FDA)-approved GUCY2C ligands can be used to test ligand replacement therapy for chemoprevention of colorectal cancer in humans. There are two GUCY2C agonists that have received FDA-approval to treat chronic idiopathic constipation and constipation-type irritable bowel syndrome, including the ST analog linaclotide (Linzess, Allergan and Ironwood Pharmaceuticals, Inc.) and the uroguanylin analog plecanatide (Trulance, Salix Pharmaceuticals) [65–67]. Both linaclotide and plecanatide activate the GUCY2C receptor and stimulate fluid secretion and cGMP production [68]. Further, both linaclotide and plecanatide have been implicated as chemoprevention agents in colorectal cancer. For example, oral linaclotide treatment reduces tumor formation in the APC^min/+ mouse model [69]. Additionally, linaclotide currently is in phase II clinical trial to explore its utility as a chemoprevention agent for colorectal tumorigenesis (Linaclotide in Treating Patients With Stages 0–3 Colorectal Cancer, NCT03796884).

Plecanatide (Salix Pharmaceuticals), another GUCY2C agonist, is structurally similar to uroguanylin, only differing by a replacement of aspartic acid with glutamic acid at the 3-position at the N-terminus, producing greater binding affinity [70]. In a mouse model of colorectal tumorigenesis, plecanatide reduced colon dysplasia by decreasing proliferation through inhibition of APC/β-catenin signaling [71]. Further, a second uroguanylin analog, dolcanatide, may hold promise as a GUCY2C agonist for chemoprevention [72]. Indeed, dolcanatide is being examined in a phase I clinical trial to test its safety for colorectal cancer prevention in healthy volunteers (NCT03300570). Together, these studies highlight an emerging strategy using GUCY2C ligand replacement as an innovative method of colorectal cancer chemoprevention.

7. Non-steroidal anti-inflammatory drugs (NSAIDs) and PDE inhibitors

Multiple epidemiological studies have correlated the long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) to a lowered risk of colorectal cancer [73,74]. Thus, the NSAID sulindac reduces both tumor count and tumor burden in a carcinogen (azoxymethane) colon tumorigenesis model, through cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2)-dependent and -independent mechanisms [75,76]. Further, the COX-2 inhibitor celecoxib decreases rectal adenomas by 28% in familial adenomatous polyposis (FAP) patients, and was approved for chemoprevention in that population. However, clinical trials of a similar COX-2 inhibitor, rofecoxib, produced unexpected cardiovascular toxicity, dampening enthusiasm for the use of NSAIDs to prevent and treat colon cancer [73]. Further studies of NSAIDs revealed the possibility of COX-1/−2-independent mechanisms of inhibition of tumor formation. For example, the COX-2 inhibitor NS-398 induces apoptosis in human cell lines regardless of change in COX-2 protein expression [77]. Further, active metabolites of sulindac, which do not inhibit COX-1/−2, increase apoptosis in cancer cell lines [78]. Thus, NSAID metabolites or derivatives that do not inhibit COX-1 or COX-2 were explored further as a safe and effective therapeutic to treat and prevent colon cancer.

Exisulind, a derivative of the NSAID sulindac, induces apoptosis in SW480 cells through PKG-mediated inhibition of oncogenic β-catenin activity. Exisulind inhibits PDE5 and PDE2, elevating intracellular cGMP levels and thereby activating PKG. PKG phosphorylates β-catenin and mediates apoptosis in these cells [79]. Furthermore, exisulind reduced tumor incidence and multiplicity in a rat model of carcinogen (azoxymethane)-induced tumorigenesis [80]. Clinically, exisulind prevented colorectal polyp formation in FAP patients over 24 months [81]. Exisulind treatment produced tumor cell apoptosis and polyp regression, but hepatic toxicity prevented FDA approval [81].

In that context, alternate cGMP elevating agents with more suitable safety profiles are being investigated. Indeed, the FDA-approved PDE5 inhibitor sildenafil [82], approved for the treatment of erectile dysfunction and pulmonary hypertension, reduces tumor formation in the APC^min/+ mouse model [69]. Further, sildenafil reduces tumor formation in mouse models of carcinogen (azoxymethane)- and inflammation (dextran sodium sulfate)-induced tumorigenesis [83]. Translating to human cancer, sildenafil treatment prevents the growth of human tumor xenografts in nude mice [82]. Moreover, population studies from national databases in Sweden suggest that the use of PDE5 inhibitors after colorectal cancer diagnosis lowers the incidence of metastasis and cancer-specific death among male patients [84]. Additionally, male patients diagnosed with benign colorectal tumors or polyps prescribed PDE5 inhibitors had a decreased risk of colorectal cancer compared to male patients not prescribed PDE5 inhibitors [85]. Further, a retrospective cohort study analyzing the Veterans Affairs Informatics and Computing Infrastructure found an 18% lower risk of colorectal cancer in male patients exposed to PDE5 inhibitors at least once [86]. Together, these data suggest that PDE5 inhibitors such as sildenafil may be a safe and effective chemoprevention for colorectal cancer. It is noteworthy that results have not been uniform. Indeed, in one population study, colorectal cancer incidence was not different in men who used PDE5 inhibitors, compared to those who did not use these agents [87]. Therefore, there is a need for further studies to validate the use of PDE5 inhibitors in colorectal cancer chemoprevention.

8. Beyond cancer prevention: GUCY2C as a biomarker

Disease staging is vital to determine therapeutic strategy in GI cancers. Traditional diagnosis by histopathological examination of lymph node tissue often leads to missed metastases and patient under-staging [88]. For example, in stage II disease, resection of tumors within the intestinal wall is often enough to cure patients. However, the National Comprehensive Cancer Network (NCCN) has determined a group of high-risk features of recurrence in stage II patients including poorly differentiated histology, a report of less than 12 lymph nodes, tumor depth defined by bowel obstruction, localized perforation, or positive margins [89,90]. Further, the American Joint Committee on Cancer (AJCC) categorizes stage II patients into IIA, IIB, and IIC by intestinal wall penetration of the primary tumor. Stage IIA (pT3N0) tumors invade through the muscularis propria into the pericolonic tissues, stage IIB (pT4aN0) tumors penetrate the surface of the visceral peritoneum, and stage IIC (pT4bN0) tumors invade or are adherent to nearby organ [90]. An analysis of the Surveillance, Epidemiology, and End Results (SEER) database revealed that the five-year overall survival rates of stages IIA, IIB, and IIC disease are 66.7%, 60.6%, and 45.7%, respectively, highlighting the heterogeneity in stage II disease [90,91]. With the presence of high-risk features, guidelines recommend that patients receive adjuvant chemotherapy [90]. Therefore, better diagnostic tools are needed to minimize diagnostic uncertainty, more accurately stage patients, and reduce disease recurrence.

The expression pattern of GUCY2C makes it a valuable biomarker of colorectal cancer metastases in extra-intestinal tissues for detection and staging. GUCY2C is expressed throughout the intestinal epithelium and in a subset of hypothalamic and midbrain neurons, but not in extraintestinal tissues such as liver and lung, which are common sites for GI cancer metastases [92–94]. Further, GUCY2C is expressed by most human colorectal primary and metastatic tumors [95,96]. While GUCY2C is not expressed by normal gastric, esophageal, and pancreatic tissues [97], it has been identified in primary and metastatic tumors from these regions [97–100]. Indeed, GUCY2C mRNA and protein are expressed in dysplasias and adenocarcinomas arising from intestinal metaplasia in esophagus and stomach [97–99,101]. Additionally, GUCY2C protein is expressed in the cytoplasmic regions of squamous cell carcinomas of esophagus [98]. Moreover, GUCY2C mRNA and protein is expressed in pancreatic cancers, likely arising through intestinal metaplasia, making it a target for GUCY2C-directed diagnostics [98,100].

Interestingly, in a blinded multicenter prospective trial, 2570 lymph nodes from 257 pN0 colorectal cancer patients were analyzed for GUCY2C mRNA. Quantitative real time polymerase chain reaction (qRT-PCR) analyses revealed that 87% of patients considered stage II by traditional histopathological staging had occult lymph node metastases by GUCY2C molecular staging. In a follow-up 24 months later, 20.9% of patients with occult metastases, while only 6.3% of patients without occult metastases, had recurrent disease [102]. Therefore, GUCY2C is being explored as a diagnostic molecular marker to improve the sensitivity and specificity of staging patients with GI cancers [103–105].

9. GUCY2C as an immunotherapy target for gastrointestinal cancers

GUCY2C is present in most human primary and metastatic colorectal tumors, as well as in approximately 60% of gastric, esophageal, and pancreatic primary and metastatic tumors [95,96,98]. Importantly, GUCY2C expression is limited to the apical brush border of the healthy intestine, which is distinctly separated from the immunological compartment in the underlying lamina propria [1]. Further, limited communication between the mucosal and systemic immune systems prevents GUCY2C-related autoimmune toxicities [106]. Therefore, ectopic expression of GUCY2C in cancers of the upper GI tract and pancreas combined with its restricted expression in the luminal compartment of healthy intestine suggest that it might be a target for immunotherapies.

9.1. GUCY2C vaccines

Cancer vaccines are a promising therapeutic strategy to prevent disease recurrence and improve survival in patients with minimal residual disease [107]. In that context, there is a need for cancer vaccines that are efficacious and do not result in autoimmunity, a hurdle that needs to be overcome when using tumor-associated antigens (TAA) [108]. Adenovirus is a common vector to deliver tumor vaccines because of the robust immune responses that they generate [109]. Thus, adenovirus-based (Ad5) cancer vaccines have been developed for GUCY2C delivery. These vaccines induce antigen-specific CD8⁺ T-cell, but not CD4⁺ T-cell or antibody, responses in syngeneic mice, associated with vaccine-induced antitumor immunity [106,110–114]. The Ad5-GUCY2C vaccine prevents the development of experimental colorectal cancer metastases, without autoimmunity, in mice after intravenous injection of GUCY2C-expressing CT26 mouse colorectal cancer cells to establish lung or liver metastases. Further, the GUCY2C adenovirus vaccine, in combination with immunological boosts of employing a variety of vectors carrying GUCY2C, is effective in reducing lung metastases without autoimmunity and increasing animal survival [106,110].

Preclinical studies demonstrating efficacy of the vaccine, with the addition of the pan HLA-DR epitope (PADRE) to boost CD4⁺ T-cell responses, formed the basis for a phase I clinical trial that explored Ad5-GUCY2C-PADRE immunization in patients with early stage (stage I-II) colorectal cancer (NCT01972737) [111,115]. Indeed, patients produced a CD8⁺ T-cell response to GUCY2C, without autoimmunity [115]. However, CD8⁺ T-cell responses were produced only in patients with low Ad5 neutralizing antibody titers (NAbs), suggesting that pre-existing Ad5-specific immunity limits the effectiveness of the vaccine.

To overcome the limitation of pre-existing Ad5 NAbs, an alternative adenoviral vector that consists of the capsid of Ad5 and fiber of the rare adenovirus serotype 35 (Ad35) was developed in conjunction with the GUCY2C antigen to generate GUCY2C-targeted antitumor immunity in a broader segment of the population [116]. Indeed, this chimeric vector resisted neutralization in Ad5-immune mice and in sera collected from colorectal cancer patients naturally exposed to Ad5 [116]. An ongoing clinical trial is examining the immunological and safety response to Ad5.F35-GUCY2C-PADRE in patients with GI cancers (NCT04111172).

9.2. GUCY2C chimeric antigen receptor (CAR)-T cells

Adoptive cell therapy (ACT) is an innovative approach to utilize a patient’s own immune system to treat metastatic cancer. The three methods widely applied include ACT using tumor-infiltrating lymphocytes, T cell receptor (TCR) modified T cells, or CAR-modified T cells. In particular, CAR-T cell therapies directed against refractory pre-B cell acute lymphoblastic leukemia and diffuse large B cell lymphoma have been approved by the FDA [117]. In CAR-T cell therapy, autologous CD8+ T cells are engineered to express a CAR specifically targeting a tumor antigen, expanded in vitro, and then infused back into patients [117]. Although currently approved CAR-T cell therapies are directed to hematological malignancies, efforts are expanding into solid tumors [117].

GUCY2C expression in the intestine is distinctly compartmentalized from the systemic immune system, making it ideal for CAR-T cells to target GUCY2C-expressing tumors without autoimmune responses in the gut. Indeed, in immunocompetent mice, CAR-T cells targeting mouse GUCY2C eliminate colorectal cancer metastases in lung without off-target toxicities [118]. Further, human GUCY2C-targeted CAR-T cells eliminate lung metastases in pre-clinical studies in syngeneic immunocompetent mice, and subcutaneous xenograft models of human colorectal cancer in immunodeficient mice [119].

9.3. GUCY2C immunotoxins

Antibodies have emerged as a mainstay of therapies that block signaling mechanisms important in tumor evolution, including oncogenic circuits and angiogenesis [120]. Additionally, antibody-drug conjugates (ADCs) are an effective method to deliver cytotoxic agents to tumors with antigen specificity. ADCs consist of monoclonal antibodies linked to antitumor cytotoxins. Antibody binding to tumor cell surface antigens results in ADC internalization and lysosomal cleavage of the linkage between antibody and cytotoxin, producing cytotoxin-driven cell death [120].

Recently, a GUCY2C-targeted ADC was developed that consists of a GUCY2C-specific antibody with a disulfide linkage (4-succinimidyloxycarbonyl-α-methyl-α−2-pyridyldithio- toluene; SMPT) to the cytotoxic ricin A chain [121]. Not only did the GUCY2C ADC kill colorectal cancer cells, but it also reduced metastatic lung tumors by 80% and improved animal survival by 25% [121]. Another GUCY2C-targeted ADC that was linked to the cytotoxic monomethyl auristatin E (MLN0264) was explored in a phase II clinical trial in patients with GUCY2C-positive gastric or gastroesophageal junction adenocarcinoma (NCT02202759). However, the trial was terminated because it did not meet the interim efficacy endpoints [122]. Similarly, a phase II clinical trial of MLN0264 in patients with advanced GUCY2C-positive pancreatic adenocarcinoma was terminated for failure to meet interim efficacy endpoints (NCT02202785) [123].

Conclusion

GUCY2C, a transmembrane receptor that regulates fluid and electrolyte secretion in the intestine, is a novel target for GI cancers [55]. The loss of its activating hormones guanylin and uroguanylin early in colorectal tumorigenesis suggests that colorectal cancer, in part, may be a disease of paracrine hormone loss silencing the receptor, and may be reversible by ligand replacement therapy [18,56,63,71]. Further, the restricted expression of GUCY2C to healthy intestinal cells and a subset of neurons, but widespread expression in primary and metastatic GI cancers, makes it an ideal diagnostic to improve the sensitivity and specificity of staging patients with GI cancers. This restricted expression also makes it a useful target for immunotherapies such as cancer vaccines and CAR-T cell therapy. In that context, there is an ongoing phase II clinical trial for the GUCY2C-targeted AD5.F35-GUCY2C-PADRE vaccine in patients with a history of GI malignancies (NCT04111172). Moreover, preclinical studies have revealed that GUCY2C targeted CAR-T cells are highly effective in eliminating colorectal cancer metastases in mice [118,119]. The potential impact of GUCY2C-targeted diagnostics and therapeutics can be appreciated by considering that, beyond colorectal cancer which has a 5 year survival of >60%, current therapies for GI cancers such as gastric, esophageal, and pancreatic cancer only provide 5-year relative survival rates of 32%, 21% and 10%, respectively [15]. These considerations highlight the considerable unmet clinical need in those diseases that might be satisfied with GUCY2C-targeted strategies.

Expert Opinion

Colorectal, and other, cancers of the GI tract are difficult to prevent and treat. GUCY2C was first associated with colorectal cancer through population studies suggesting an inverse relationship worldwide between colorectal cancer prevalence and enterotoxigenic Escherichia coli (ETEC) infections [54]. Since then, there has been a focus on identifying the role of GUCY2C signaling in prevention of colorectal cancer. Promising research in the use of GUCY2C signaling directly through ligand supplementation as chemoprevention for colon cancer has emerged [63,69]. Supplementing preclinical laboratory studies, clinical trials assessing the pharmacodynamic (PD) effects of linaclotide on cGMP levels, based on biopsy samples of adenomas or resected colorectal adenocarcinomas in patients with stages 0–3 colorectal cancer are ongoing (Linaclotide in Treating Patients With Stages 0–3 Colorectal Cancer, NCT03796884). Further, preclinical studies are exploring the utility of other GUCY2C agonists, plecanatide and dolcanatide, in mouse models of colorectal cancer.

Although population and preclinical studies suggest that PDE5 inhibitors are useful to prevent colorectal cancer, results have not been uniform. For example, Soriana et al published a population study in which they surprisingly identified that colorectal cancer incidence did not differ between users and non-users of PDE5 inhibitors [87]. In contrast, Sutton et al conducted a retrospective cohort study analyzing the Veterans Affairs Informatics and Computing Infrastructure, and found an association of an 18% lowered risk of colorectal cancer in male patients exposed at least once to PDE5 inhibitors [86]. Conflicting studies like these highlight the need for further exploration of the use of PDE5 inhibitors to prevent colorectal cancer. Although there are many studies implicating PDE inhibitors in preventing colorectal cancer, a major hurdle could be safety. However, these considerations may be mitigated by using the FDA-approved PDE5 inhibitor, sildenafil, since there are no long-term risks associated with its current use for erectile dysfunction [124].

The use of GUCY2C as a diagnostic and therapeutic target is not exclusively limited to colorectal cancer. It can be translated to other tumors arising in the GI tract, such as gastric, esophageal, and pancreatic cancers, which may express GUCY2C [97–101]. Thus, metastases in these diseases may be treated using GUCY2C-targeted CAR-T cells. In that context, CAR-T cell products have been approved for mantle cell and large B-cell lymphomas as well as acute lymphoblastic leukemia [117]. Although CAR-T cell therapies have yet to be approved for solid tumors, they are the subjects of intense research efforts. In that context, preclinical studies show that GUCY2C CAR-T cells are both safe and effective in treating colorectal metastatic tumors [118,119]. The expression of GUCY2C in other cancers of the GI tract suggests that CAR-T cell therapy also could be effective for those tumor types. Further, preclinical studies of the Ad5.F35-GUCY2C-PADRE vaccine suggest that it could be highly effective for the secondary prevention of metastases in patients with colorectal cancers at risk for recurrent disease [116]. It is anticipated that these strategies will be extended to other GI cancers.

Not only has GUCY2C been implicated as a target for chemoprevention and treatment of GI cancers, but it has also been recognized as a novel biomarker. Its expression in lymph nodes exclusively in metastatic disease makes it a specific biomarker to improve the accuracy of staging patients with colorectal cancer. Additionally, its presence in some gastric, esophageal, and pancreatic tumors, but not in normal tissue from those organs, suggests that it might be a novel marker for the diagnosis of the earliest stages of transformation in these tissues. Together, the study of GUCY2C suggests a promising route to prevent and treat not only colorectal cancers, but also other GI cancers with substantial unmet clinical needs.

Article Highlights.

• The GUCY2C signaling axis regulates fluid and electrolyte secretion in the intestine.

• Loss of GUCY2C signaling occurs early and almost universally in colorectal cancer through aberrant APC-mediated suppression of guanylin hormone expression.

• An ongoing clinical trial explores the reactivation of the GUCY2C receptor as a method for colorectal cancer chemoprevention through the use of the FDA-approved GUCY2C agonist, linaclotide.

• Preclinical studies of cyclic GMP elevating agents, such as the FDA-approved PDE5 inhibitor sildenafil, suggest they may be promising for chemoprevention.

• The expression of GUCY2C in cancers of the colorectum, esophagus, stomach, and pancreas allows its use as a diagnostic biomarker for cancer staging.

• GUCY2C is a target for immunotherapies, such as vaccines, CAR-T cells, and immunotoxins. An ongoing phase II clinical trial is testing the efficacy of a GUCY2C-targeted vaccine to prevent secondary metastatic disease in patients with cancers arising in the GI tract.

Abbreviations:

APC

adenomatous polyposis coli

BMP

bone morphogenetic protein

cAMP

cyclic adenosine monophosphate

CFTR

cystic fibrosis transmembrane conductance regulator

cGMP

cyclic guanosine monophosphate

EGF

epidermal growth factor

ETEC

enterotoxigenic Escherichia coli

FAP

familial adenomatous polyposis

GI

gastrointestinal

GTP

guanosine triphosphate

GUCY2C

guanylyl cyclase C

ISC

intestinal stem cell

NHE3

Na⁺/H⁺ exchanger III

NSAID

non-steroidal anti-inflammatory drug

PDE3

phosphodiesterase 3

PDE5

phosphodiesterase 5

PKA

protein kinase A

PKGII

protein kinase G II

qRT-PCR

quantitative real time polymerase chain reaction

ST

bacterial heat-stable enterotoxin

TA

transit amplifying

TAA

tumor-associated antigens

TCF

T-cell factor

5’-AMP

5’-adenosine monophosphate

5’-GMP

5’-guanosine monophosphate