Guanylyl Cyclase C Expression Research Articles

Abstract 2636: Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer

Abstract Background: Amatoxin-based antibody drug conjugates (so called ATAC®s) constitute a new class of antibody-drug conjugates (ADCs) which utilize amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits cellular transcription. In the current study, in vitro and in vivo data of an ATAC® targeting GCC (guanylyl cyclase C) are presented. GCC is a cell surface receptor expressed in >95% of colorectal cancer, and in approximately 65% of esophageal, gastric, and pancreatic tumors. In healthy conditions GCC expression is restricted to the gastrointestinal tract, and more specific to the apical brush border of the intestinal epithelium (luminal site). Thus, GCC in healthy tissue is not exposed to the circulation but upon tumor progression in GI malignancies it becomes accessible for i.v. injected targeted therapeutics. This tumor-specific accessibility for drugs in circulation makes GCC a highly attractive target for ATAC®s. Material and Methods: ATAC®s: Cysteine-reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ATAC®s with a DAR of 2.0. Animal models: Subcutaneous (s.c.) mouse xenograft models with the GCC overexpressing cell line HEK293-GUCY2C mono2 or colon cancer PDX models (Charles River) were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in NMRI nude mice and non-human primates (NHP). Results: All anti-GCC ATAC®s with amanitin-linker derivatives, optimized for use in solid tumors, showed favorable in vitro cytotoxicity with picomolar activity on GCC+ cell lines and no cytotoxic activity on target-negative cells. In mouse xenograft models, the optimized anti-GCC ATAC®s caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Even in colorectal cancer PDX models, anti- GCC ATAC®s led to a substantial anti-tumor effect. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ATAC®s tested in mice differed between 7.5 and 50 mg/kg depending on linker chemistry. Safety profiling in cynomolgus monkeys revealed good tolerability for all selected ATAC®s. Hematology and clinical chemistry parameters were unaffected apart from a moderate and transient increase in liver enzymes. Conclusions: Targeted cytotoxic drug delivery to GCC+ cell lines was achieved by using anti-GCC ATAC®s optimized for the use in solid tumors. The mode of action of the payload amanitin led to a pronounced anti-tumor effect in vitro and in vivo in PDX models with good tolerability in non-human primates. The use of anti-GCC ATAC®s with their novel mode of action in the therapy of GCC positive cancers, such as colorectal cancer represents a promising approach in cancer therapy. Citation Format: Alexandra Braun, Andreas Pelz, Franziska Ebeling, Kristin Decker, Anikó Pálfi, Michael Kulke, Andreas Pahl, Torsten Hechler. Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2636.

Evaluation of TAK-264, an Antibody-Drug Conjugate in Pancreatic Cancer Cell Lines and Patient-Derived Xenograft Models.

Antibody-drug conjugates (ADCs) are an emerging technology consisting of an antibody, linker, and toxic agent, which have the potential to offer a targeted therapeutic approach. A novel target recently explored for the treatment of pancreatic cancer is guanylyl cyclase C (GCC). The objective of this study was to determine the anti-tumorigenic activity of TAK-264, an investigational ADC consisting of an antibody targeting GCC linked to a monomethyl auristatin E payload via a peptide linker. The antiproliferative effects of TAK-264 assessed in a panel of eleven pancreatic cancer cell lines. Additionally, ten unique pancreatic ductal adenocarcinoma cancer patient-derived xenograft models were treated with TAK-264 and the efficacy was determined. Baseline levels of GCC were analyzed on PDX models and cell lines. Immunoblotting was performed to evaluate the effects of TAK-264 on downstream effectors. GCC protein expression was analyzed by immunoblotting in both normal and tumor tissue; marked increase in GCC expression was observed in tumor tissue. The in vitro experiments demonstrated a range of responses to TAK-264. Eight of the ten PDAC PDX models treated with TAK-264 demonstrated a statistically significant tumor growth inhibition. Immunoblotting demonstrated an increase in phosphorylated-HistoneH3 in both responsive and less responsive cell lines and PDAC PDX models treated with TAK-264. There was no correlation between baseline levels of GCC and response in either PDX or cell line models. TAK-264 has shown suppression activity in pancreatic cancer cell lines and in pancreatic PDX models. These findings support further investigation of ADC targeting GCC.

Abstract 3916: Relationship of guanylyl cyclase C (GCC) expression and efficacy of TAK-164, a GCC-targeted antibody-drug conjugate in a panel of 68 subcutaneous HuPrime colorectal cancer PDX models

Abstract Guanylyl cyclase C (GCC) is a cell surface protein expressed in more than 95% of metastatic colorectal cancers and in the majority of gastric and pancreatic cancers, making GCC an attractive antibody-drug conjugate (ADC) target. TAK-164 is an ADC comprised of a full-length, fully human IgG1 monoclonal antibody (mAb) directed toward the extracellular domain of GCC. The mAb is conjugated using ImmunoGen's peptide-linked indolinobenzodiazepine DNA alkylator DGN549. Previously presented preclinical studies using TAK-164 have demonstrated in vivo efficacy in a select number of GCC-expressing tumor models following a single administration of TAK-164. Here, we describe the selection of an appropriate dose for broad testing in a panel of patient-derived xenograft models of colorectal cancer. The efficacy data generated following a single administration of 15 µg/kg TAK-164 (payload concentration) were compared with molecular features that could aid in understanding drivers of TAK-164 activity, including GCC expression by immunohistochemistry (IHC). Primary colorectal cancer models were derived from colorectal adenocarcinomas and implanted in immunocompromised mice. Once tumors reached a stratified mean volume of 150-250 mm3, animals were randomized into treatment groups (n = 1/group) and dosing was initiated on Day 0 of the study. There were three tumor groups (Vehicle treated, TAK-164 treated, and naïve), which were used to characterize genomic and molecular features of the model. The planned study endpoint was 60 days or until a humane endpoint was reached. For all models included in the study, an IHC assay to detect GCC was used to explore the relationship between GCC expression and time to rise (TTR), a preclinical tumor progression measurement. TTR is the Delta duration in days on study for each PDX model (TAK-164 - Vehicle) to reach 1000 mm3 tumor volume. In the present study, a mixed effects Cox regression analysis, using a continuous or binary stratification of GCC IHC expression, resulted in a statistically significant (p<0.05) increase in TTR in TAK-164−treated mice with higher expression levels of GCC. Ongoing analysis aims to better understand additional molecular drivers of TAK-164 treatment that may be useful for patient enrichment strategies in the clinic. TAK-164 is scheduled to enter phase I evaluation in 2018 in GCC-positive colorectal cancer and other GI malignancies. Citation Format: Erik M. Koenig, Cong Li, Huyuan Yang, Andy Zhu, Pooja Shah, Kazuho Nishimura, Bret Bannerman, Mengkun zhang, Bradley Stringer, Brittany Bahamon, O. Petter Veiby, Adnan Abu-Yousif. Relationship of guanylyl cyclase C (GCC) expression and efficacy of TAK-164, a GCC-targeted antibody-drug conjugate in a panel of 68 subcutaneous HuPrime colorectal cancer PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3916.

A monomethyl auristatin E-conjugated antibody to guanylyl cyclase C is cytotoxic to target-expressing cells in vitro and in vivo.

Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.

Abstract B050: Evaluation of TAK-264, a novel antibody-drug conjugate in pancreatic cancer cell lines and patient-derived xenograft models

Abstract Background: Antibody-drug conjugates (ADCs) are an emerging technology consisting of antibody, linker, and toxic agent, which have the potential to offer a more targeted therapeutic approach than standard chemotherapy regimens. ADCs selectively bind to antigens expressed in tumor environments. An emerging target for the treatment of pancreatic cancer is guanylyl cyclase C (GCC). GCC is a transmembrane G protein receptor found on the apical surface of intestinal enterocytes. It plays an important role in GI homeostasis. GCC is highly expressed in various colorectal carcinomas as well as adenocarcinomas of the upper GI tract. The objective of this study was to determine the antitumorigenic activity of TAK-264, an investigational ADC that targets GCC. Experimental Procedures: The antiproliferative effects of TAK-264 were assessed in a panel of eleven pancreatic cancer cell lines with various molecular backgrounds. Additionally, ten unique pancreatic ductal adenocarcinoma (PDAC) cancer patient-derived xenograft (PDX) models were treated with TAK-264 and the efficacy was determined. Tumor size was evaluated twice per week by caliper measurements. Sensitivity to TAK-264 was defined based on tumor growth inhibition that was statistically significant when compared to the vehicle control. Baseline levels of GCC were analyzed by IHC/immunoblotting and RT-PCR on PDX models and cell lines. Immunoblotting was performed to evaluate the effects of TAK-264 on downstream effectors. Results: GCC protein expression was analyzed by immunoblotting in normal versus tumor tissue; a marked increase in GCC expression was observed in tumor tissue when compared to matching normal tissue. The in vitro experiments demonstrated a range of responses to TAK-264 in an SRB assay. Eight of the ten PDAC PDX models demonstrated a statistically significant tumor growth inhibition when compared to the vehicle control. Immunoblotting demonstrated an increase in phosphorylated-Histone-H3 in two cell lines and in the PDAC PDX models treated with TAK-264, indicating a DNA damage response. The analysis of GCC protein expression in normal versus tumor tissue has shown a marked increase in GCC expression in tumor tissue when compared to matching normal tissue. There was no correlation between baseline levels of GCC and response to TAK-264 in either PDX or cell line models. Conclusions: TAK-264, an ADC targeting GCC, has good growth suppression activity in pancreatic cancer cell lines and in pancreatic PDX models. These findings support a hypothesis that further investigation of ADC targeting GCC may lead to novel therapeutic modalities for pancreatic cancer. Citation Format: Anna R. Schreiber, Anna Nguyen, Stacey M. Bagby, Betelehem Yacob, Kevin Quackenbush, Joe L. Guy, Thomas Crowell, Bradley Stringer, Hadi Danaee, Thea Kalebic, Wells A. Messersmith, John J. Arcaroli, Todd M. Pitts. Evaluation of TAK-264, a novel antibody-drug conjugate in pancreatic cancer cell lines and patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B050.

Expression of guanylyl cyclase C in tissue samples and the circulation of rectal cancer patients.

Guanylyl cyclase C (GCC) is a transmembrane surface receptor restricted to intestinal epithelial cells, from the duodenum to the rectum. We compared GCC expression in tumors and normal rectal tissues, and investigated the relation between GCC expression and metastasis and long-term survival of rectal cancer patients. Based on the UICC classification, 42 rectal cancer patients in this study were classified as stage I, 48 patients as stage II, and 90 patients as stage III. Overexpression of GCC was observed in 80 rectal tumors as compared to matched normal tissues, where no strong staining of GCC was observed. An association between GCC mRNA in the circulation and tumor emboli in vessels, CK20 mRNA, distant organ metastasis, and survival status was observed in 100 rectal cancer patients. Univariate Cox regression analysis indicated that tumor emboli in vessels, lymph node metastasis, mesenteric root lymph node metastasis and GCC mRNA correlated with 5-year disease-free survival (DFS); while lymph node metastasis, GCC mRNA, and CK20 mRNA strongly correlated with 5-year overall survival (OS). In a multivariate Cox regression model, GCC mRNA level and mesenteric root lymph node metastasis associated with DFS, while GCC mRNA levels associated with OS. Quantification of GCC expression in circulation is a valuable biomarker for assessing tumor burden and predicting outcome in rectal cancer patients.

A Pilot Study of CK19, CK20 and GCC mRNA in the Peripheral Blood as a Colorectal Cancer Biomarker Panel.

Colorectal cancer remains one of the major cancer- related deaths despite progress in the treatment during past decades. Detection of disease at earlier stages reduces its mortality. The aim of current study was to investigate expression of Cytokeratin 19 (CK19), Cytokeratin 20 (CK20) and Guanylyl Cyclase C (GCC) mRNA in peripheral blood of non- metastatic colorectal cancer patients which may result into introducing of an early detection test. 25 patients with colorectal cancer and 25 healthy controls were recruited. Blood was obtained from all individuals. Expression of CK19 and CK20 and GCC mRNA and 18SrRNA (as reference gene) were determined based on real- time RT-PCR on total RNA from blood. CK19, CK20 and GCC expression had been detected in 68%, 76% & 52% of patient group, respectively, which was higher than healthy group, with 8%, 32% and 0% expression, respectively (p<0.05). CK20 was over-expressed 8- fold more in patients compared to controls. Similar result was found for CK19 with 4- fold over- expression. Sensitivity and specificity of combination of markers were 88% and 68%, respectively. Current data suggest that the detection of CK20 & CK19 as relative sensitive markers may become a valuable tool for primary diagnosis of colorectal cancer in early stages. GCC could be considered as a specific tumor marker for detection of colorectal cancer. Higher expression of these markers in patients may be considered as a relative good tool for the diagnosis of disease in non- metastatic stages.

Abstract 4649: Using pharmacokinetic/efficacy modeling to identify the optimal schedule for MLN0264, an anti- guanylyl cyclase C (GCC) antibody-drug conjugate, in a range of xenograft models

Abstract MLN0264 is an investigational antibody-drug conjugate (ADC) that consists of the human anti-guanylyl cyclase C (GCC) antibody linked to a microtubule-disrupting agent (monomethyl auristatin). As ADCs have a very long clearance half-life, the potential exists for a highly infrequent dosing schedule. A quantitative understanding of the relationship between exposure and preclinical antitumor biological activity is thus applied to support dose schedule selection in the clinic. In this study, we develop a pharmacokinetic/efficacy (PK/E) relationship in xenograft models to evaluate the predictive contributions of exposure and xenograft characteristics to MLN0264 biological activity. Single dose pharmacokinetic (PK) data were obtained for a range of time points, and a linear two-compartment PK model was built. Xenograft biological activity studies were conducted in which MLN0264 was administered at various dose levels and dosing schedules to mice bearing one of six different xenograft models. We used multiple linear regression and tumor dynamic modeling to understand the factors contributing to the biological activity. First, the growth rate of each tumor under control and treatment conditions was established by fitting the biological activity data within the dosing period to an exponential growth function. Then, we assessed the relationship of AUC to antitumor biological activity. Within each xenograft model, AUC was strongly correlated with biological activity across a range of schedules (R2&amp;gt;0.9, p&amp;lt;0.03, for all models). AUC-proportional biological activity was confirmed using dynamic modeling with direct drug effect to simulate the tumor dynamics for different dosing schedules. Although biological activity within a xenograft model was strongly correlated with exposure, when the data from different xenograft models were pooled together, the correlation was weaker (R2= 0.30, p= 0.0004). This result suggests that, although schedule is not a major determinant of biological activity, other xenograft-specific factors may be contributing to biological activity. To test the contribution of some possible factors, we used multiple linear regression to determine which covariates (such as GCC expression level and baseline growth rate of xenograft models) are predictive of biological activity. The results suggest baseline growth rate does not correlate with biological activity while GCC expression is weakly correlated. This finding formed the basis for the development of a mechanistic model of GCC expression and biological activity. Taken together, this work demonstrates the use of a PK/E framework to identify the scheduling effect for a first-in-man protocol for an ADC. We have also demonstrated that this PK/E framework can be further leveraged to assess the contribution of other potential predictors of ADC biological activity in xenograft models. Citation Format: Shu-Wen Teng, Christopher Zopf, Johnny Yang, Brad Stringer, Julie Zhang, Wen Chyi Shyu, Arijit Chakravarty, Petter Veiby, Jerome Mettetal. Using pharmacokinetic/efficacy modeling to identify the optimal schedule for MLN0264, an anti- guanylyl cyclase C (GCC) antibody-drug conjugate, in a range of xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4649. doi:10.1158/1538-7445.AM2014-4649

Abstract PR12: MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC), demonstrates antitumor activity alone and in combination with gemcitabine in human pancreatic cancer xenograft models expressing GCC.

Abstract Background: MLN0264 consists of a fully human anti-GCC monoclonal antibody linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker (MMAE and linker technology licensed from Seattle Genetics). The transmembrane cell surface receptor GCC is expressed by ∼95% of primary and metastatic colorectal cancer (mCRC) tumors. MLN0264 has shown selective binding and antitumor activity in mouse xenograft models of mCRC expressing GCC, and is currently being investigated in a phase 1 study in patients with advanced gastrointestinal malignancies. GCC is also expressed in subsets of pancreatic cancers. Further to the findings in mCRC, we investigated GCC expression in human pancreatic tumors and evaluated MLN0264 activity in mouse xenograft models of GCC-expressing human pancreatic cancer. Methods: GCC expression in multiple human pancreatic cancer samples including tissue microarrays (TMAs) was evaluated by immunohistochemistry (IHC). For in vivo studies, 7 mouse xenograft primary human tumor explant (PHTX) models of pancreatic cancer were developed, including tumor tissue from patients with wild-type and mutant KRAS. Animals were treated when the tumor reached ∼230 mm3. In single-agent studies, animals were administered vehicle, MMAE 0.135 mg/kg once weekly (QW), or MLN0264 3.75 or 7.5 mg/kg QW. In combination studies, animals received vehicle, or MLN0264 7.5 mg/kg QW alone or in combination with gemcitabine 15 or 20 mg/kg twice weekly (BIW), or gemcitabine 15 mg/kg on days 1, 3 each week. Average tumor volume was determined at multiple time points following the start of treatment using vernier callipers. Results: In the GCC-expressing PHTX-249 mouse xenograft model (KRAS mutant G12), single-agent MLN0264 showed significant tumor growth inhibition (TGI) versus vehicle or free MMAE by day 21, with the 7.5 mg/kg dose significantly better than 3.75 mg/kg by day 20-22. Similarly, in the GCC-expressing PHTX-215 model (KRAS wild-type), MLN0264 7.5 mg/kg resulted in significantly greater TGI (79%) versus free MMAE or MLN0264 3.75 mg/kg by day 22, including some tumor regression. Across the 7 models (variable apical GCC expression; KRAS wild-type and mutant), TGI ranged from 24% (p=0.17) to 79% (p&amp;lt;0.001) with single-agent MLN0264. In the PHTX-249 model, MLN0264 7.5 mg/kg plus gemcitabine 15/20 mg/kg BIW or 15 mg/kg, days 1, 3, showed significantly greater TGI than either agent alone. At day 20-21, TGI was 46-47% for single-agent MLN0264, 66-79% for gemcitabine 15 mg/kg BIW and gemcitabine days 1, 3, and 84-88% for the respective combinations; 3 of 7 animals in the latter combination group had a smaller tumor volume at day 20 versus day 0 (TGI, 93%). Conclusions: These findings indicate that MLN0264 has antitumor activity as a single agent and in combination with gemcitabine in GCC-expressing pancreatic cancer xenograft models, and support clinical evaluation of MLN0264 in patients with pancreatic cancer. Data from additional models will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR12. Citation Format: Julie Zhang, Melissa Gallery, Tim Wyant, Brad Stringer, Mark Manfredi, Hadi Danaee, Petter Veiby. MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC), demonstrates antitumor activity alone and in combination with gemcitabine in human pancreatic cancer xenograft models expressing GCC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR12.

Targeting the GCC endocrine signaling axis to reduce appetite in obesity

In light of our discovery that the transmembrane receptor guanylyl cyclase C (GCC) regulates appetite through a novel gut‐brain endocrine axis, we aimed to further define hypothalamic GCC expression and, moreover, determine the consequence of obesity to this GCC signaling axis. Nuclei were microdissected from mouse hypothalamus and GCC expression was quantified by RT‐PCR. Mouse brains were also cryosectioned and hypothalamic slices were immunostained for GCC. Further, hypothalamus and intestine were harvested from lean and obese mice, and GCC and GCC ligand expression was quantified by RT‐PCR, immunoblot, and immunofluorescence. Our studies revealed the suppression of intestinal ligand expression with the concomitant overexpression of hypothalamic GCC in obese mice. Further, our studies suggested that GCC was enriched in the arcuate nucleus, a key regulator of appetite. Our results suggest a corruption of the GCC axis in obesity, which in part reflects an endocrine deficiency of GCC ligands that could be therapeutically targeted to supersensitized receptors by hormone replacement. Thus, GCC represents a new target to develop anti‐obesity pharmacotherapies against this accelerating pandemic. This research was supported by the Thomas Jefferson University Doctoral Student Research Fellowship.

Association of peripheral blood guanylyl cyclase C (GCC) expressions with prognostic parameters and response to therapy in patients with colorectal cancer.

508 Background: Guanylyl cyclase C (GCC) is expressed exclusively in normal intestinal mucosal cells, primary and metastatic colorectal cancers (CRC). The aim of this study was to determine the possible association between the GCC expressions in peripheral blood, prognostic parameters and response to chemotherapy in CRC patients. Methods: Forty-nine metastatic CRC patients and 41 healthy controls with similar age and sex were included to this study. Peripheral blood GCC expressions are measured by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Results: No GCC expression was measured in healthy controls but GCC expressions of the patients were detectable. Although there was a significant reduction in GCC expressions in 30 patients with regression (from 5.46 ± 4.12 to 0.06 ± 0.03, p &lt; 0.0001), marked increase in GCC expressions was observed in 19 patients with progression following chemotherapy (from 0.43 ± 0.19 to 1.38 ± 0.52, p = 0.0174). Significant correlation was found between the GCC expressions and carbohydrate antigen 19-9 (CA19-9) levels (p = 0.0041) in 30 patients with regression before chemotherapy. Marked correlation was also detected between the GCC expressions and carcinoembryonic antigen (CEA) levels (p = 0.0072) in 19 patients with progression before chemotherapy. Conclusions: The results of the present study suggest that peripheral blood GCC expressions along with CEA and CA19-9 can be used to determine the early respose to chemotherapy in patients with metastatic CRC. These findings imply that higher expression of GCC in peripheral blood seems to be an indicator of good therapeutic response to chemotherapy and remission. Monitoring the peripheral blood GCC expressions may allow employing different treatment options to metastatic CRC patients.

Peripheral blood guanylyl cyclase c (GCC) expressions are associated with prognostic parameters and response to therapy in colorectal cancer patients

Guanylyl cyclase C (GCC) is expressed exclusively in normal intestinal mucosal cells, primary and metastatic colorectal cancers (CRC). The aim of this study was to determine the possible association between the GCC expressions in peripheral blood, prognostic parameters and response to chemotherapy in CRC patients. Fourty-nine metastatic CRC patients and 41 healthy controls with similar age and sex were included to this study. Peripheral blood GCC expressions are measured by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Interstingly, no GCC expression was measured in healthy controls but GCC expressions of the patients were detectable. Although there was a significant reduction in GCC expressions in 30 patients with regression (from 5.46 ± 4.12 to 0.06 ± 0.03, p < 0.0001), marked increase in GCC expressions was observed in 19 patients with progression following chemotherapy (from 0.43 ± 0.19 to 1.38 ± 0.52, p = 0.0174). Significant correlation was found between the GCC expressions and carbohydrate antigen 19-9 (CA19-9) levels (p = 0.0041) in 30 patients with regression before chemotherapy. Marked correlation was also detected between the GCC expressions and carcinoembryonic antigen (CEA) levels (p = 0.0072) in 19 patients with progression before chemotherapy. The results of the present study suggest that peripheral blood GCC expressions along with CEA and CA19-9 can be used to determine the early respose to chemotherapy in patients with metastatic CRC. These findings imply that higher expression of GCC in peripheral blood seems to be an indicator of good therapeutic response to chemotherapy and remission. Monitoring the peripheral blood GCC expressions may allow employing different treatment options to metastatic CRC patients.

GCC expression in lymph nodes (LNs) as a significant determinant of recurrence in stage II colon cancer (CC) patients (pts).

369 Background: A multi-center prospectively specified retrospective study Validating Indicators to Associate Recurrence (VITAR) is assessing the relationship between guanylyl cyclase C (GCC) gene expression in formalin fixed LNs and recurrence risk in stage II CC pts not treated with adjuvant chemotherapy. Here we report the preplanned initial analysis performed with 241 pts. Methods: GCC mRNA was quantified by RT-qPCR using FFPE LNs tissues from untreated stage II CC pts diagnosed from 1999-2006 with at least 10 LN examined blinded to clinical outcomes. Cox regression models examined the relationship between GCC nodal status and the prespecified primary endpoint of recurrence risk. Results: Twenty-ninepts (12%) had a disease recurrence or cancer death, median follow-up was 60 months and median LNs examined was 15. The ratio of the number of GCC+ LNs over the total number of informative LNs (LNR) significantly predicted higher recurrence risk for 84 pts classified as high risk (HR, 2.38; p=0.02). The estimated 5-yr recurrence rates were 10% and 27% for the low and high risk group, respectively. After adjusting for age, T stage, number of LNs assessed, and MMR status, the significant association remained (HR, 2.61; 95% CI, 1.17-5.83; p=0.02). In a subset of 181 pts with negative margin, T3 tumor only and ≥12 LN examined, the GCC LNR had a HR for recurrence of 5.06 (95% CI 1.61-15.91, p=0.003), translating into 5-yr recurrence rates of 4% among low risk pts and 27% for the high-risk group. Conclusions: Our results suggest that GCC expression in LNs is a significant determinant of recurrence in appropriately staged CC pts not treated with adjuvant chemotherapy. The validation component of the study is ongoing. [Table: see text] [Table: see text]

Expression of guanylyl cyclase-c and caudal type homeobox transcription factor 2 in human gastric cancer and precursor lesions

To investigate the expression of guanylyl cyclase-C (GC-C) and caudal type homeobox transcription factor 2 (CDX2) in human gastric mucosa at different stages and the significance thereof. An Immunofluorescence method was used to detect the expression of GC-C and CDX-2 in 23 specimens of gastric carcinoma and matching noncancerous tissues. Western blotting was used to detect the protein expression of GC-C and CDX2 in the gastric carcinoma tissues and matching noncancerous tissues too. The GC-C and CDX2 expression rates were 39.1% and 39.1% respectively in the intestinal metaplasia specimens, 55.6% and 55.6% respectively in the dysplasia specimens, and 56.7 % and 60.0% in the gastric carcinoma specimens, all significantly higher than those in the normal mucosa specimens (all P = 0.000) without significant differences in the expression of GC-C and CDX-2 among the 3 pathological groups. The GC-C and CDX-2 expression was positively correlated with Lauren classification, The expression levels of GC-C and CDX-2 were significantly higher in the intestinal-type than in the diffuse-type gastric carcinoma (P < 0.05). The GC-C expression was positively correlated with the expression of CDX-2 in intestinal metaplasia and gastric carcinoma. Ectopic expression of GC-C and CDX2 in human gastric mucosa may play an important role in the carcinogenesis of intestinal-type gastric carcinoma. Detection of GC-C and CDX2 helps diagnose gastric carcinoma and precursor lesions.

Sex Modulates Intestinal Transformation by the Tumor‐Suppressor GCC

There is an established relationship between sex and colorectal tumorigenesis, and the annual age-adjusted incidence of colorectal cancer in the United States is higher in men than in women. In addition, there are sex differences regarding the anatomic location and clinicopathological characteristics of tumors. Moreover, hormone replacement therapy (HRT) in postmenopausal women reduces the risk of colorectal cancer by 30–40%. However, while ovarian hormones mitigate colorectal cancer risk by modifying genetic and environmental determinants, the precise mechanisms remain undefined. In that regard, the most commonly lost gene products in intestinal neoplasia include guanylin and uroguanylin, paracrine hormones for guanylyl cyclase C (GCC), an emergent tumor-suppressor regulating crypt hyperplasia, proliferation, and the cell cycle. Of note, the molecular intersection between intestinal paracrine and systemic sex hormones opposing intestinal neoplasia has not been explored. Here, the impact of GCC on tumorigenesis was examined in mice carrying mutations in Apc (Apc Min/+) or exposed to azoxymethane (AOM), models of genetic and chemical intestinal carcinogenesis. Proliferation of intestinal epithelial cells was quantified employing molecular markers of the cell cycle. While elimination of GCC expression increased tumor burden in Apc Min/+ and AOM-treated mice, females exhibited only approximately 50% of the burden observed in males. Of note, elimination of GCC expression promoted epithelial cell proliferation in males but not in females. These observations reveal an intersection between sex and the GCC-based paracrine hormone axis, converging at restriction of crypt cell proliferation. They suggest that the invariable loss of guanylin and uroguanylin and the resultant downstream effects on tumorigenesis are mitigated by sex. In the context of the established association between HRT, menopause, and the risk of colon cancer, and the inhibition by estrogen of cell cycle regulators, it is presumed that hormonal components of the ovarian axis mediate these protective effects. This previously unappreciated synergy between sex and the GCC signaling axis suggests a unique opportunity for cancer prevention and treatment. Indeed, while expression of guanylin and uroguanylin is invariably lost early along the continuum of neoplastic transformation, GCC is universally overexpressed by human colorectal tumors. In the context of the standard of care in which hormone deficiencies are treated by replacement, the present observations underscore the potential for combining systemic sex and GCC paracrine hormone supplementation for targeted prevention and therapy in colorectal cancer.

A prospective multicenter study of guanylyl cyclase C (GCC), quantified by the reverse transcriptase-polymerase chain reaction (qRT-PCR), as a prognostic marker of occult metastases in lymph nodes of pN0 colorectal cancer patients

11011 Background: Patients with pN0 colorectal cancer have a ∼25% risk of disease recurrence reflecting, in part, under-diagnosis of lymph node metastases at staging. Improved methods that predict recurrence would identify patients who could benefit from adjuvant chemotherapy. GCC, expressed selectively by intestinal cells and universally in colorectal tumors, is a marker whose detection in lymph nodes could enhance staging. Methods: At staging, 356 patients with pN0 colorectal cancer from 9 hospitals were enrolled. Fresh lymph nodes >5 mm were bisected for histopathology and GCC qRT-PCR. Three hundred patients provided lymph nodes, of which 261 provided tissues with sufficient mRNA for analysis. Categorical GCC expression, normalized to beta-actin, was estimated in 2,452 lymph nodes from these patients, who were followed to assess time to recurrence. Results: Median follow-up of patients was 45 months (range 1–75 months). Of the 261 patients, 13% (34 patients) had lymph nodes free of occult metastases by GCC qRT-PCR and all but 2 remained free of disease (5.8% raw recurrence rate). Conversely, 20.7% (47 patients) with lymph nodes containing occult metastases by GCC qRT-PCR developed recurrent disease (p=0.029, log-rank test). In multivariable Cox models, controlling for T stage, tumor location, lymphovascular invasion, and tumor differentiation, occult metastases in lymph nodes by GCC qRT-PCR was the most powerful independent predictor of recurrence (adjusted Hazard Ratio=4.51, p=0.039, 95%CI=1.07, 18.90). Conclusions: GCC qRT-PCR identifies occult metastases in lymph nodes that independently predict time to recurrence in pN0 colorectal cancer patients. Thus, GCC may serve as a prognostic and predictive marker, identifying pN0 patients at minimum risk for disease recurrence and, conversely, who might benefit from adjuvant chemotherapy, respectively. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Targeted Diagnostics and Therapeutics, Inc. Targeted Diagnostics and Therapeutics, Inc. Targeted Diagnostics and Therapeutics, Inc.

Homeostatic Control of the Crypt-Villus Axis by the Bacterial Enterotoxin Receptor Guanylyl Cyclase C Restricts the Proliferating Compartment in Intestine

Guanylyl cyclase C (GC-C), the receptor for diarrheagenic enterotoxins and the paracrine ligands guanylin and uroguanylin, regulates intestinal secretion. Beyond volume homeostasis, its importance in modulating cancer cell proliferation and its uniform dysregulation early in colon carcinogenesis, reflecting loss of ligand expression, suggests a role for GC-C in organizing the crypt-villus axis. Here, eliminating GC-C expression in mice 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 cells, but not enteroendocrine cells. GC-C signaling regulated proliferation by restricting the cell cycle at the G(1)/S transition. Moreover, crypt expansion in GC-C(-/-) mice was associated with adaptive increases in cell migration and apoptosis. Reciprocal alterations in proliferation and differentiation resulting in expansion associated with adaptive responses in migration and apoptosis suggest that GC-C coordinates component processes maintaining homeostasis of the crypt progenitor compartment. In the context of uniform loss of GC-C signaling during tumorigenesis, dysregulation of those homeostatic processes may contribute to mechanisms underlying colon cancer.

A Validated Quantitative Assay to Detect Occult Micrometastases by Reverse Transcriptase-Polymerase Chain Reaction of Guanylyl Cyclase C in Patients with Colorectal Cancer

Guanylyl cyclase C (GCC), a receptor for bacterial diarrheagenic enterotoxins, may be a prognostic and predictive marker to detect occult micrometastases in patients undergoing staging for colorectal cancer. However, quantification of GCC expression in tissues by the quantitative reverse transcription-PCR (qRT-PCR) has not undergone analytic and clinicopathologic validation. A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC. GCC qRT-PCR exhibited reaction efficiencies >92%, coefficients of variations <5%, linearity >6 orders of magnitude, and a limit of quantification of >25 copies of GCC cRNA. This assay confirmed that GCC mRNA was overexpressed by colorectal tumors from 41 patients, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses obtained with 164 lymph nodes from patients free of cancer and 15 nodes harboring metastases established a threshold for metastatic disease of approximately 200 GCC mRNA copies/mug total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (approximately 21%) nodes free of disease by histopathology from 6 of 23 (26%) patients, suggesting the presence of occult micrometastases. Quantifying GCC mRNA in tissues by RT-PCR employing external calibration standards is analytically robust and reproducible, with high clinicopathologic sensitivity and specificity. This validated assay is being applied to approximately 10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.

Bile Acids Induce Ectopic Expression of Intestinal Guanylyl Cyclase C Through Nuclear Factor-κB and Cdx2 in Human Esophageal Cells

Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids. Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis. GC-C mRNA and protein were ectopically expressed in approximately 80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor kappaB (NF-kappaB). Furthermore, deoxycholate increased NF-kappaB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-kappaB subunit p50. Moreover, a dominant negative construct for NF-kappaB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter. Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-kappaB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C.

Lack of guanylyl cyclase C, the receptor for Escherichia coli heat‐stable enterotoxin, results in reduced polyp formation and increased apoptosis in the multiple intestinal neoplasia (Min) mouse model

Guanylyl cyclase C (GC-C), a transmembrane receptor for bacterial heat-stable enterotoxin and the mammalian peptides guanylin and uroguanylin, mediates intestinal ion secretion and affects intestinal cell growth via cyclic GMP signaling. In intestinal tumors, GC-C expression is maintained while guanylin and uroguanylin expression is lost, suggesting a role for GC-C activation in tumor formation or growth. We show by in situ hybridization that GC-C expression is retained in adenomas from multiple intestinal neoplasia (Apc(Min/+)) mice. In order to determine the in vivo role of GC-C in intestinal tumorigenesis, we generated Apc(Min/+) mice homozygous for a targeted deletion of the gene encoding GC-C and hypothesized that these mice would have increased tumor multiplicity and size compared to wild-type Apc(Min/+) mice on the same genetic background. In contrast, the absence of GC-C resulted in a reduction of median polyp number by 55%. There was no change in the median diameter of polyps, suggesting no effect on tumor growth. Somatic loss of the wild-type Apc allele, an initiating event in intestinal tumorigenesis, also occurred in polyps from GC-C-deficient Apc(Min/+) mice. We have found increased levels of apoptosis as well as increased caspase-3 and caspase-7 gene expression in the intestines of GC-C-deficient Apc(Min/+) mice compared with Apc(Min/+) mice. We propose that these alterations are a possible compensatory mechanism by which loss of GC-C signaling also affects tumorigenesis.