Internal Medicine
Clinical Pharmacology

Guanylyl Cyclase C is a Novel Tumor Susceptibility Gene in Colorectal Cancer. The most commonly lost gene products in colorectal carcinogenesis include guanylin and uroguanylin, the endogenous ligands for guanylyl cyclase C (GCC, GUCY2C), the intestinal receptor for diarrheagenic bacterial enterotoxins. The significance of loss of GCC ligands early in intestinal tumorigenesis remains obscure because (patho)physiological roles of GCC signaling in intestine, beyond regulation of intestinal fluid and electrolyte homeostasis, are poorly defined. Recently, signaling by GCC and its downstream effector, cyclic GMP (cGMP) has emerged as a principal regulator of proliferation in human colon cancer cells. In that context, differentiated enterocytes in villi exhibit higher guanylin expression and ligand-dependent cGMP accumulation compared to proliferating progenitor cells in crypts. These observations suggest that GCC signaling may regulate the renewal of the intestinal epithelium by restricting the proliferating compartment and promoting the transition from proliferation to differentiation along the crypt to villus axis. Here, the role of GCC in regulating normal enterocyte dynamics along the crypt-villus axis and the corruption of those mechanisms in tumorigenesis were explored. Elimination of GCC expression in mice (GCC-/-) increased crypt length along a decreasing rostral-caudal gradient by disrupting component homeostatic processes. Crypt expansion reflected hyperplasia of the proliferating compartment with reciprocal increases in rapidly cycling progenitor cells and reductions in differentiated cells of the secretory lineage, including Paneth and goblet, but not enteroendocrine, cells. In close agreement, induction of GCC signaling in mucosal sheets ex vivo and intestinal cells in vitro inhibited proliferation by activating cGMP-dependent protein kinase and delaying the cell cycle at the G1/S transition. Crypt hyperplasia in GCC-/- mice was associated with compensatory increases in cell migration and apoptosis. This novel role in restricting the proliferating compartment and organizing the crypt-villus axis suggests that dysregulation of GCC, reflecting loss of endogenous ligands, might contribute to tumorigenesis by potentiating hyperproliferation which, in turn, may amplify disruption of genomic integrity. Indeed, in colons of mice carrying mutations in Apc (ApcMin/+) or exposed to the carcinogen azoxymethane, elimination of GCC increased tumor initiation by corrupting genomic integrity in the context of disabled compensatory apoptosis. Separately, deletion of GCC increased tumor growth by releasing a restriction on the cell cycle normally constraining epithelial cell proliferation. This previously unrecognized role of GCC as a tumor suppressor by restricting proliferation and maintaining genomic integrity suggests that receptor dysregulation reflecting ligand insufficiency is a key event during early colorectal tumorigenesis. Together with the uniform over-expression of GCC in human tumors, and the standard of care in which hormone deficiencies are treated by replacement therapy, the role of GCC as a tumor suppressor underscores the potential of oral administration of GCC ligands for targeted prevention and therapy of colorectal cancer.

Guanylyl Cyclase C Establishes Lineage Dependence in Colorectal Cancer. An evolving paradigm expanding the genetic basis of cancer suggests that developmental programs indelibly imprint restricted repertoires of homeostasis, forming the substrate for lineage-dependent tumor induction by deregulation of tissue-specific survival pathways. Guanylyl cyclase C (GCC), the intestinal receptor for the paracrine hormones guanylin and uroguanylin whose early loss characterizes transformation, has emerged as a component of developmental programs organizing spatiotemporal patterning along the crypt-surface axis whose loss promotes tumorigenesis through hyperproliferation and genetic instability. This project focuses on defining the role of GCC as a switch for lineage-dependent tumorigenesis whose deregulation reprograms survival circuits essential for regenerative homeostasis and universally required for neoplastic transformation. Indeed, eliminating GCC in mice expands proliferating crypts, accelerating the cell cycle by regulating mediators of the G1/S transition. Proliferative induction is coupled with metabolic remodeling, with an increase in aerobic glycolysis and a reciprocal reduction in mitochondrial biogenesis and oxidative phosphorylation, recapitulating human tumors. Conversely, GCC signaling restores survival circuits in colon cancer cells, decelerating the cell cycle and switching from glycolytic to mitochondrial ATP production, recapitulating normal enterocytes. Coordination of survival circuits by GCC is orchestrated through the oncogene AKT, whose inhibition mimics and activation eliminates GCC regulation of proliferation and metabolism. GCC modulates AKT through the tumor suppressor PTEN, whose silencing eliminates GCC regulation of AKT, proliferation and metabolism. Thus, disruption of developmentally restricted signaling by GCC reflecting loss of paracrine hormones induces maladaptive oncogene-dependent survival pathways, normally essential for homeostasis, whose tissue-specific deregulation contributes to lineage addiction in intestinal tumorigenesis. With the role of guanylin and uroguanylin loss in transformation, universal over-expression of GUCY2C by colorectal tumors offers a unique therapeutic opportunity for cancer prevention through oral hormone replacement therapy.

The Tumor Suppressor Guanylyl Cyclase C Directs Epithelial-Mesenchymal Interactions Opposing Stromal Reactivity through TGF-B1-Mediated Signaling. The epithelium of the intestine undergoes continuous homeostatic regeneration mediated by proliferation, migration, and differentiation, an imbalance of which may be one mechanism contributing to colorectal carcinogenesis. Guanylyl cyclase C (GCC), the receptor for the endogenous paracrine hormones guanylin and uroguanylin and the exogenous bacterial diarrheagenic heat-stable enterotoxins (ST), is exclusively expressed in the apical membranes of enterocytes and supplies intracellular cyclic GMP (cGMP), maintaining intestinal fluid and electrolyte balance. Recently, GCC emerged as a key tumor susceptibility gene whose elimination produces unrestricted epithelial cell proliferation, metabolic remodeling. and genomic instability, the combination of which accelerates tumorigenesis in mouse models of chemical and genetic intestinal carcinogenesis. Additionally, guanylin and uroguanylin are the most frequently lost gene products in colorectal tumorigenesis in animals and humans, and the expression of these hormones is invariably attenuated at the earliest stages of neoplastic development. Taken together, these observations suggest a novel paracrine hormone hypothesis for colorectal cancer, wherein GCC coordinates key processes underlying epithelial homeostasis and its dysregulation. Beyond cell autonomous processes within epithelial cells, non-autonomous interactions with underlying mesenchymal elements maintain this homeostasis through cell-cell contact and paracrine signaling. Activated fibroblasts, a principal component of the mesenchymal microenvironment, regulates key homeostatic processes, including deposition of extracellular matrix (ECM), epithelial cell proliferation migration, and angiogenesis, contributing to dynamic modifications of the interstitium driving cancer progression. In the context of the role of guanylyl cyclase and cGMP in regulating stromal remodeling in other systems, the working hypothesis suggests that GCC-cGMP signaling regulates epithelial-mesenchymal interactions in intestine and loss of that signaling contributes to the development of a reactive stromal niche and neoplastic transformation. These studies reveal a previously unappreciated role for GCC and cGMP signaling in directing epithelial cell remodeling of the stromal compartment in intestine.

Cancer Mucosa Antigens as Immunotherapeutic Targets for Metastatic Tumors. Immunotherapy for human cancers is hindered, in part, by a lack of suitable target antigens. This is particularly relevant in tumors derived from mucosal tissues such as colorectal cancer, in which antigens that are sufficiently immunogenic, tumor-restricted and shared among patients are lacking, and for which conventional therapeutics are poorly efficacious. We have explored a novel class of tumor-associated antigens fulfilling these criteria by exploiting immune compartmentalization, which restricts cross-talk between systemic and mucosal immune compartments. This compartmentalization limits systemic tolerance to mucosa-restricted self-antigens and shields mucosa from systemic autoimmune responses. Thus, a novel paradigm suggests that targeting self-antigens expressed by normal mucosal tissues and by derivative neoplasms should permit effective immunotherapy against systemic metastases, without inducing autoimmunity in normal mucosa. We have explored targeting the first of these antigens, termed cancer mucosa antigens (CMAs), in animal models of metastatic cancer. Viral vector vaccines were generated containing guanylyl cyclase C (GCC), expressed in normal intestinal epithelium and in all primary and metastatic human colorectal cancer (CRC) specimens. Immunization elicited CD8+ T, but not CD4+ T or B, cell responses in multiple strains of mice. Moreover, responses effectively prevented development of lung and liver CRC metastases and treated established lung CRC metastases. This occurred in the absence of autoimmunity against normal GCC-expressing intestinal tissue. These results suggest the utility of GCC-specific immunotherapy for gastrointestinal malignancies, as well as the potential for CMA-targeted immunotherapy for malignancies of other mucosae such as oral, respiratory, mammary, and urogenital tissues, to treat head and neck, lung, breast, and bladder cancers, respectively.

Targeting Gastrointestinal Malignancies with Ligand-Directed Heterofunctional Nanodevices. Using guanylyl cyclase C (GCC) as a specific molecular target, the goal of this project is to fabricate a heterofunctional targeted-delivery nano-device that specifically delivers both imaging and therapeutic agents to colorectal cancer metastases, but not normal cells. GCC is the intestinal receptor for bacterial diarrheagenic STs and the endogenous ligands guanylin and uroguanylin. GCC is selectively expressed in apical membranes of intestinal cells and resides outside mucosal tight junctions, sequestered in the lumenal compartment of the intestine, inaccessible to the systemic vascular compartment. That radiolabeled ST administered intravenously specifically traffics only to colon cancer xenografts, but not to extra-intestinal tissues or normal intestine, in mice in vivo underscores the integrity of this compartmentalization. Moreover, GCC is over-expressed by human colorectal cancer cells, and GCC expression has been detected in all primary and metastatic colorectal tumors examined. Over-expression of GCC by colorectal cancer cells and its anatomical compartmentalization confined to mucosa, but accessible to the systemic vasculature in metastases, suggests its utility as a biomarker for delivering imaging and therapeutic agents in vivo. Here, specific targeting of metastatic tumors will be achieved by conjugating multifunctional payloads to a GCC ligand, the bacterial heat-stable enterotoxin (ST). The heterofunctional targeting agent will be fabricated by conjugating ST and the therapeutic or imaging moieties to biotin, followed by docking to the functional ligand binding sites of avidin. In this configuration, biotinylated ST occupies one of the four binding sites on avidin while the 3 remaining binding sites can be occupied by biotinylated radionuclide for imaging or biotinylated cytotoxic compounds for therapy. ST has been biotinylated and this conjugate docked to tetrameric avidin. Further, ST-conjugated avidin specifically binds to GCC expressed by colorectal cancer cells in vitro. Selective internalization of this ST-avidin conjugate by GCC-expressing colorectal cancer cells currently is being examined. In that context, ligand/receptor-based molecular targeting provides a highly selective delivery mechanism and ST-targeted nanodevices are poised to significantly advance colorectal cancer diagnostics, staging, management, and ultimately patient survival.

Guanylyl Cyclase C mRNA in Lymph Nodes Predicts Time to Recurrence and Disease-Free Survival in pN0 Colorectal Cancer. Histopathological examination of lymph nodes from colorectal cancer (CRC) patients provides a measure of systemic tumor progression and prognosis. Remarkably, ~30% of patients with lymph node (LN)-negative (pN0; stage I and II) colorectal cancer develop recurrent disease within 5 years of presumptively curative resection. This rate of recurrence underscores the heterogeneity of pN0 patients, and the limitations of current paradigms to detect metastases resulting in the under-staging of CRC patients. Consequently, a variety of sensitive immuno- and nucleic acid-based molecular amplification assays have evolved to more accurately detect occult metastases. One such approach, Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) that detects mRNA targets, offers the promise of a clinically significant prognostic and predictive assay that more accurately stratifies pN0 patients, which should better enable matching patients with appropriate postoperative treatment to improve disease outcome. Guanylyl cyclase C (GCC) is an apical transmembrane receptor that regulates fluid and electrolyte homeostasis whose expression is restricted to the intestinal mucosa and which continues to be expressed universally by primary and metastatic colorectal tumors. A target-specific qRT-PCR assay was developed and analytically validated employing an external calibration standard to quantify GCC mRNA. This assay revealed that GCC mRNA was over-expressed in colorectal tumors compared to normal mucosa, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses of LN from patients with inflammatory or non-neoplastic disease (true negative) and those harboring colorectal metastases detected by histopathology (true positive) established a threshold of detection of occult metastases of ~200 GCC mRNA copies/µg total RNA with a sensitivity of 93% and a specificity of 97%. This analytically-validated assay was employed to evaluate 257 patients with pN0 colorectal cancer who were enrolled prospectively at nine centers and provided 2,570 fresh lymph nodes for histopathology and qRT-PCR. Patients were followed for a median of 45 months (range: 1-75) to estimate time to recurrence and disease-free survival. Thirty-two (12.5%) patients had lymph nodes negative by GCC qRT-PCR [pN0(mol-)], and all but two remained free of disease during follow-up (recurrence rate 6.3% [95% Confidence Interval (CI), 0.8-20.8%]). Conversely, 225 (87.5%) patients had lymph nodes positive by GCC qRT-PCR [pN0(mol+)], and 47 (20.9% [CI, 15.8-26.8%]) developed recurrent disease (p=0.006). Multivariate analyses revealed that GCC expression in lymph nodes was the most powerful independent prognostic marker. Patients who were pN0(mol+) exhibited an increased hazard of earlier time to recurrence (adjusted hazard ratio 4.42 [CI, 1.05-18.53]; p=0.042) and disease-related events associated with reduced disease-free survival (adjusted hazard ratio 3.10 [CI, 1.09-8.82]; p=0.034). Thus, the positivity of histologically negative lymph nodes by GCC qRT-PCR is independently associated with time to recurrence and disease-free survival in patients with pN0 colorectal cancer. These observations demonstrate that GCC is an indicator of occult LN metastases, identifying pN0 patients at high risk for disease recurrence who might benefit from adjuvant chemotherapy.