- Email: [email protected]
Mo1915 Novel Wnt-Targeted Therapy Inhibitors for Hepatocellular Carcinoma
AGA Abstracts
Mo1913
Mo1915
Influence of a Dipeptidyl Peptidase-4 Inhibitor on Intestinal Neoplasia Formation in APC MIN/+ Mice Fed a High Fat Diet Kaori Fujiwara, Takuya Inoue, Naoki Yorifuji, Munetaka Iguchi, Taisuke Sakanaka, Ken Narabayashi, Toshihiko Okada, Sadaharu Nouda, Kazuki Kakimoto, Kumi Ishida, Ken Kawakami, Yosuke Abe, Toshihisa Takeuchi, Eiji Umegaki, Kazuhide Higuchi
Novel Wnt-Targeted Therapy Inhibitors for Hepatocellular Carcinoma Stefania Asciutti, Elisabetta Ferretti, Sara Valloscuro, Maira M. Pires, Laura Llacuna, Davide Esposito, Paride Liscio, Antonio Macchiarulo, Emidio Camaioni, Roberto Pellicciari, Stuart A. Aaronson
Background: The association between type 2 diabetes mellitus and intestinal neoplasia has been shown epidemiologically. It is also well known that a high fat diet (HFD) promotes insulin resistance which is a risk factor for intestinal neoplasia. Dipeptidyl peptidase-4 (DPP4) inhibitors are used clinically in diabetes therapy to prolong the effects of glucagon-like peptide-1 (GLP-1). However, because another derivative from proglucagon, the intestinotrophic hormone GLP-2, is also a substrate for DPP-4, DPP-4 inhibition may enhance intestinal carcinogenesis by increasing endogenous active GLP-2. We therefore conducted this study to investigate the influence of a DPP-4 inhibitor on intestinal tumorigenesis in Apc Min/+ mice fed a HFD. Material and Methods: Segmental differences in DPP-4, 8, and 9 mRNA expression and DPP enzyme activity were determined in the mice. The DPP-4 inhibitor, sitagliptin (0.3, 1 or 3 mg/kg), was given orally. Mucosal DPP activity and GLP concentrations were measured 24 hr after administration. Six-week-old male Apc Min/+ mice were randomized to either a HFD (60 kcal fat) group or control diet (10 kcal fat) group for 9 weeks. Each group was divided into two groups, which were either treated or not treated with sitagliptin (3 mg/kg/day, i.g.). The mice were sacrificed 9 weeks after the initiation of treatment. The small intestine was coiled up into a Swiss roll, which was used to determine the number, size, and localization of tumors. Results: DPP-4 mRNA was expressed at high levels in the jejunum and ileum, with high DPP-4 activity also being detected. Oral sitagliptin caused a dose-dependent suppression in mucosal DPP-4 activity. Although the mucosal concentration of total GLP-2 was not affected, the concentration of GLP-1(7-36) was significantly elevated in the mucosa of the ileum. From seven to eight weeks after the initiation of sitagliptin treatment, bloody and inconsistent stools were observed mainly in the control diet and sitagliptin-treated mice. These mice also showed significantly lower body weight gains. In the control diet group, daily treatment with sitagliptin caused a significant increase in the number of intestinal tumors compared with mice not treated with sitagliptin. Daily treatment with sitagliptin suppressed the number of intestinal tumors in the HFD group, although this change was not statistically significant. Conclusion: This study demonstrated that chronic treatment with sitagliptin increased endogenous mucosal GLPs in the intestine and promoted intestinal tumorigenesis in lean mice, whereas sitagliptin treatment tended to suppress tumorigenesis in mice fed a HFD. Because a HFD promotes insulin resistance and intestinal neoplasia, DPP-4 inhibition may also be useful as an agent for preventing cancer in obese or diabetic patients.
Hepatocellular carcinoma (HCC) is an aggressive malignancy with dismal prognosis, and limited therapeutical options. The multikinase inhibitor sorafenib is the only currently approved systemic therapy for patients with advanced disease. However, its effect of on patient survival is limited. Wnt/βcatenin signaling is frequently activated in HCC cells and tumors. In the past years many groups have been focusing on targeting this pathway in several cancers, including HCC. So far, tankyrase (TNKS) and porcupine inhibitors have been the most promising classes of inhibitors; however, the compounds currently under investigation still have limited clinical applications. Methods: Axin1 mutated PLC-PRF-5 & SNU475, βcatenin mutated HepG2 & SNU398, autocrine Wnt activated HuH7 & Hep3B, and Wnt negative SNU449 & SNU387 HCC lines, were used. Cell cycle profile and apoptosis were analyzed by FACS. Colony formation assays were quantified at 2 weeks using crystal violet staining. Epithelial to mesenchymal transition (EMT) was measured by assessing CDH1, SNAI1, TWIST1, SNAI2, CDH2, & VIM expression levels by qPCR. Invasion was measured in matrigel coated Boyden chambers. Results: By high-throughput screening we discovered a family of novel Wnt inhibitors belonging to the TNKS inhibitor class but structurally different from any of the currently well characterized TNKS inhibitors. Based on the inhibitory effect on TCF-reporter activity, we selected the three most active and specific compounds (inactive towards other reporters i.e. NOTCH or TGFβ). We demonstrated that treatment with these inhibitors leads to massive growth arrest followed by apoptosis of Wnt activated lines. These effects were dose-dependently related to the downregulation levels of Wnt signaling. Of note, our inhibitors were biologically significantly more active than the prototypical TNKS inhibitors XAV939 and IWR1. Interestingly, we established for the first time that inhibiting TNKS-1&2 antagonizes Wnt signaling in HCC tumor lines harboring biallelic non-functional AXIN1 mutations, arguing against the current model (Huang et al, Nature 2009) for the mechanism of action for this class of inhibitors. Intriguingly, we observed loss of EMT and decreased invasion after 24h of treatment in Wnt activated lines. We further characterized SNAI1 among the most affected transcriptional regulator of this biological process and, by in silico analysis, identified conserved binding sites for LEF/TCF in SNAI1 regulatory region. We have generated a SNAI1-EGFP reporter construct and currently monitoring SNAI1 expression in Wnt-modulated xenografts during tumor formation and invasion in vivo. Conclusions: We have generated novel and potent Wnt small molecule inhibitors, which, by affecting the EMT signature and invasive HCC phenotype, can potentially lead to improved therapy for patients with Wnt activated HCC.
Mo1914
Mo1916
Thiazolidinediones but Not Metformin Significantly Reduce Colorectal Neoplastic Lesions in Diabetics At Risk for Colorectal Neoplasia Martin Tobi, Yu-Xiao Yang, Fadi Antaki, Mary Rambus, Michael J. Lawson
EMAST Within Stage II/III Colorectal Cancers Reduces the Effectiveness of Adjuvant 5-Fluorouracil on Patient Survival Yashushi Hamaya, Carla Guarinos, Stephanie Tseng-Rogenski, Rodrigo Jover, John M. Carethers
The evidence that metformin as diabetic treatment is associated with reduced colorectal neoplasia(CRN) is conflicted. The metabolic syndrome (MS) is associated with excess CRN and thus medication that treats the diabetic component of MS would be expected to reduce CRN. Methods: We investigated 117 diabetics at above average CRN risk identified by screening by a risk questionnaire, for CRN. The patients were enrolled to participate in prospective CRC screening studies at the Detroit VAMC, taking metformin, thiazolidinediones and other medications at the time of enrollment and subsequent colonoscopy. We compared the CRN outcome in enrollees not taking the medications of interest. We ascertained demographics, presence of other medications, nutritional parameters (total caloric, protein and vitamin intake, BMI, alcohol intake, fried food, etc). Primary outcomes were CRN (adenomas & CRC) found on colonoscopy. Secondary endpoints were: adenoma-carcinoma biomarker binding in body fluids and mucosal field effects (FE) using the Adnab-9 antibody. Results: There were no statistically significant differences between medication groups with respect to other characteristics that included age, diabetes duration , BMI, HbA1c, GFR. There was a significant correlation between Adnab-9 effluent binding and right-sided CRN (OR 2.1;CI 1.02-4.0;p<0.03) or any neoplasia (OR1.8;CI 1.1-3-3;p<0.05) but not with medications. Adenoma-carcinoma biomarker binding in stool, or mucosa was not statistically significantly different between the groups, as for other nutritional parameters. Thiazolidinedione intake was associated with less neoplasia (26.2% not taking had neoplasia versus 9.33% patients taking [OR3.45;CI1.22-9.74p<0.04]). There was no significant reduction effect of metformin on neoplasia ASA, NSAID, sulfonylureas; statins;insulin, and multivitamins showed no significant CRN differences. Conclusions: Unlike thiazolidinediones , metformin had no effect on of CRN reduction in diabetic patients. The adenoma reduction observed in patients on thiazolidinediones may be explained by via a reduction of the de novo inflammatory pathway by thiazolidinedione therapy, a PPARγ ligand, known to have efficacy in reducing inflammation in ulcerative colitis patients where risk of CRC is increased (Lewis JD et al. AJG 96;3323,2001) and not found to increase neoplasia risk (Ibid GE 2008;135:1914). This medication's popularity, with a possible double benefit for this population in terms of diabetic control and reduction of CRC risk, is liable to increase after increased risk of treatment CVS complications was recently dispelled. No differences between the adenoma-carcinoma sequence biomarker parameters and individual drug intake was found, supporting the notion that thiazolidinediones reduce colorectal neoplasia in this population by suppressing the inflammatory de novo pathway.
AGA Abstracts
Background and Aims: Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5FU) within DNA and subsequent cell death was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSβ (hMSH2-hMSH3 heterodimer) MMR complexes were present, was intermediate with hMutSα alone, and least with hMutSβ alone. Additionally, the effectiveness of adjuvant 5FU in patients with stage II/III CRC is dependent on a functional MMR system. We tested the hypothesis that stage II/III CRC patients whose cancers demonstrate EMAST (signifying hMSH3 and thus hMutSβ dysfunction) may not have the same response to adjuvant 5FU as EMAST-negative (fully functional MMR) CRC patients on survival. Methods: We analyzed 197 stage II/III sporadic colorectal cancers for which we had 5FU treatment and survival data. Archival DNA was extracted and analyzed for microsatellite instability (MSI: >2 of 5 NCI-recommended markers mutated) and EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). We generated Kaplan-Meier survival curves and utilized the log-rank test to determine statistical differences between groups. Results: We identified 52 (26%) MSI cancers and 93 (47%) EMAST-positive cancers, with 38 cancers showing both MSI and EMAST. Sixty-one patients (31%) received adjuvant 5FU chemotherapy, and median follow-up for all patients was 45 months. After excluding patients with MSI cancers that could confound the analysis, patients with EMAST-positive colon cancer did not improve survival with adjuvant 5FU (P>0.05) whereas patients with EMASTnegative colon cancer improved survival with adjuvant 5FU treatment (P=0.024). Including patients with MSI cancers did not alter the outcome for patients with EMAST-positive or EMAST-negative cancers treated with 5FU, and 5FU did not improve survival for patients with MSI cancers. Conclusions: There is improved survival in stage II/III CRC patients with EMAST-negative cancers after adjuvant 5FU-based chemotherapy that does not extend to patients with EMAST-positive cancers. The presence of hMSH3 (and thus hMutSβ function) is likely additive with hMutSα function within EMAST-negative tumors to improve the effectiveness of adjuvant 5FU for patient survival. Patients with MSI cancers do not improve their survival with adjuvant 5FU as previously reported. Determining the EMAST (and MSI) status of CRC patients may predict their response to 5FU-based chemotherapy. The presence of EMAST (and MSI) is one reason for the reduced effectiveness of adjuvant 5FU therapy in CRC patients.
S-690
Mo1913
Mo1915
Influence of a Dipeptidyl Peptidase-4 Inhibitor on Intestinal Neoplasia Formation in APC MIN/+ Mice Fed a High Fat Diet Kaori Fujiwara, Takuya Inoue, Naoki Yorifuji, Munetaka Iguchi, Taisuke Sakanaka, Ken Narabayashi, Toshihiko Okada, Sadaharu Nouda, Kazuki Kakimoto, Kumi Ishida, Ken Kawakami, Yosuke Abe, Toshihisa Takeuchi, Eiji Umegaki, Kazuhide Higuchi
Novel Wnt-Targeted Therapy Inhibitors for Hepatocellular Carcinoma Stefania Asciutti, Elisabetta Ferretti, Sara Valloscuro, Maira M. Pires, Laura Llacuna, Davide Esposito, Paride Liscio, Antonio Macchiarulo, Emidio Camaioni, Roberto Pellicciari, Stuart A. Aaronson
Background: The association between type 2 diabetes mellitus and intestinal neoplasia has been shown epidemiologically. It is also well known that a high fat diet (HFD) promotes insulin resistance which is a risk factor for intestinal neoplasia. Dipeptidyl peptidase-4 (DPP4) inhibitors are used clinically in diabetes therapy to prolong the effects of glucagon-like peptide-1 (GLP-1). However, because another derivative from proglucagon, the intestinotrophic hormone GLP-2, is also a substrate for DPP-4, DPP-4 inhibition may enhance intestinal carcinogenesis by increasing endogenous active GLP-2. We therefore conducted this study to investigate the influence of a DPP-4 inhibitor on intestinal tumorigenesis in Apc Min/+ mice fed a HFD. Material and Methods: Segmental differences in DPP-4, 8, and 9 mRNA expression and DPP enzyme activity were determined in the mice. The DPP-4 inhibitor, sitagliptin (0.3, 1 or 3 mg/kg), was given orally. Mucosal DPP activity and GLP concentrations were measured 24 hr after administration. Six-week-old male Apc Min/+ mice were randomized to either a HFD (60 kcal fat) group or control diet (10 kcal fat) group for 9 weeks. Each group was divided into two groups, which were either treated or not treated with sitagliptin (3 mg/kg/day, i.g.). The mice were sacrificed 9 weeks after the initiation of treatment. The small intestine was coiled up into a Swiss roll, which was used to determine the number, size, and localization of tumors. Results: DPP-4 mRNA was expressed at high levels in the jejunum and ileum, with high DPP-4 activity also being detected. Oral sitagliptin caused a dose-dependent suppression in mucosal DPP-4 activity. Although the mucosal concentration of total GLP-2 was not affected, the concentration of GLP-1(7-36) was significantly elevated in the mucosa of the ileum. From seven to eight weeks after the initiation of sitagliptin treatment, bloody and inconsistent stools were observed mainly in the control diet and sitagliptin-treated mice. These mice also showed significantly lower body weight gains. In the control diet group, daily treatment with sitagliptin caused a significant increase in the number of intestinal tumors compared with mice not treated with sitagliptin. Daily treatment with sitagliptin suppressed the number of intestinal tumors in the HFD group, although this change was not statistically significant. Conclusion: This study demonstrated that chronic treatment with sitagliptin increased endogenous mucosal GLPs in the intestine and promoted intestinal tumorigenesis in lean mice, whereas sitagliptin treatment tended to suppress tumorigenesis in mice fed a HFD. Because a HFD promotes insulin resistance and intestinal neoplasia, DPP-4 inhibition may also be useful as an agent for preventing cancer in obese or diabetic patients.
Hepatocellular carcinoma (HCC) is an aggressive malignancy with dismal prognosis, and limited therapeutical options. The multikinase inhibitor sorafenib is the only currently approved systemic therapy for patients with advanced disease. However, its effect of on patient survival is limited. Wnt/βcatenin signaling is frequently activated in HCC cells and tumors. In the past years many groups have been focusing on targeting this pathway in several cancers, including HCC. So far, tankyrase (TNKS) and porcupine inhibitors have been the most promising classes of inhibitors; however, the compounds currently under investigation still have limited clinical applications. Methods: Axin1 mutated PLC-PRF-5 & SNU475, βcatenin mutated HepG2 & SNU398, autocrine Wnt activated HuH7 & Hep3B, and Wnt negative SNU449 & SNU387 HCC lines, were used. Cell cycle profile and apoptosis were analyzed by FACS. Colony formation assays were quantified at 2 weeks using crystal violet staining. Epithelial to mesenchymal transition (EMT) was measured by assessing CDH1, SNAI1, TWIST1, SNAI2, CDH2, & VIM expression levels by qPCR. Invasion was measured in matrigel coated Boyden chambers. Results: By high-throughput screening we discovered a family of novel Wnt inhibitors belonging to the TNKS inhibitor class but structurally different from any of the currently well characterized TNKS inhibitors. Based on the inhibitory effect on TCF-reporter activity, we selected the three most active and specific compounds (inactive towards other reporters i.e. NOTCH or TGFβ). We demonstrated that treatment with these inhibitors leads to massive growth arrest followed by apoptosis of Wnt activated lines. These effects were dose-dependently related to the downregulation levels of Wnt signaling. Of note, our inhibitors were biologically significantly more active than the prototypical TNKS inhibitors XAV939 and IWR1. Interestingly, we established for the first time that inhibiting TNKS-1&2 antagonizes Wnt signaling in HCC tumor lines harboring biallelic non-functional AXIN1 mutations, arguing against the current model (Huang et al, Nature 2009) for the mechanism of action for this class of inhibitors. Intriguingly, we observed loss of EMT and decreased invasion after 24h of treatment in Wnt activated lines. We further characterized SNAI1 among the most affected transcriptional regulator of this biological process and, by in silico analysis, identified conserved binding sites for LEF/TCF in SNAI1 regulatory region. We have generated a SNAI1-EGFP reporter construct and currently monitoring SNAI1 expression in Wnt-modulated xenografts during tumor formation and invasion in vivo. Conclusions: We have generated novel and potent Wnt small molecule inhibitors, which, by affecting the EMT signature and invasive HCC phenotype, can potentially lead to improved therapy for patients with Wnt activated HCC.
Mo1914
Mo1916
Thiazolidinediones but Not Metformin Significantly Reduce Colorectal Neoplastic Lesions in Diabetics At Risk for Colorectal Neoplasia Martin Tobi, Yu-Xiao Yang, Fadi Antaki, Mary Rambus, Michael J. Lawson
EMAST Within Stage II/III Colorectal Cancers Reduces the Effectiveness of Adjuvant 5-Fluorouracil on Patient Survival Yashushi Hamaya, Carla Guarinos, Stephanie Tseng-Rogenski, Rodrigo Jover, John M. Carethers
The evidence that metformin as diabetic treatment is associated with reduced colorectal neoplasia(CRN) is conflicted. The metabolic syndrome (MS) is associated with excess CRN and thus medication that treats the diabetic component of MS would be expected to reduce CRN. Methods: We investigated 117 diabetics at above average CRN risk identified by screening by a risk questionnaire, for CRN. The patients were enrolled to participate in prospective CRC screening studies at the Detroit VAMC, taking metformin, thiazolidinediones and other medications at the time of enrollment and subsequent colonoscopy. We compared the CRN outcome in enrollees not taking the medications of interest. We ascertained demographics, presence of other medications, nutritional parameters (total caloric, protein and vitamin intake, BMI, alcohol intake, fried food, etc). Primary outcomes were CRN (adenomas & CRC) found on colonoscopy. Secondary endpoints were: adenoma-carcinoma biomarker binding in body fluids and mucosal field effects (FE) using the Adnab-9 antibody. Results: There were no statistically significant differences between medication groups with respect to other characteristics that included age, diabetes duration , BMI, HbA1c, GFR. There was a significant correlation between Adnab-9 effluent binding and right-sided CRN (OR 2.1;CI 1.02-4.0;p<0.03) or any neoplasia (OR1.8;CI 1.1-3-3;p<0.05) but not with medications. Adenoma-carcinoma biomarker binding in stool, or mucosa was not statistically significantly different between the groups, as for other nutritional parameters. Thiazolidinedione intake was associated with less neoplasia (26.2% not taking had neoplasia versus 9.33% patients taking [OR3.45;CI1.22-9.74p<0.04]). There was no significant reduction effect of metformin on neoplasia ASA, NSAID, sulfonylureas; statins;insulin, and multivitamins showed no significant CRN differences. Conclusions: Unlike thiazolidinediones , metformin had no effect on of CRN reduction in diabetic patients. The adenoma reduction observed in patients on thiazolidinediones may be explained by via a reduction of the de novo inflammatory pathway by thiazolidinedione therapy, a PPARγ ligand, known to have efficacy in reducing inflammation in ulcerative colitis patients where risk of CRC is increased (Lewis JD et al. AJG 96;3323,2001) and not found to increase neoplasia risk (Ibid GE 2008;135:1914). This medication's popularity, with a possible double benefit for this population in terms of diabetic control and reduction of CRC risk, is liable to increase after increased risk of treatment CVS complications was recently dispelled. No differences between the adenoma-carcinoma sequence biomarker parameters and individual drug intake was found, supporting the notion that thiazolidinediones reduce colorectal neoplasia in this population by suppressing the inflammatory de novo pathway.
AGA Abstracts
Background and Aims: Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5FU) within DNA and subsequent cell death was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSβ (hMSH2-hMSH3 heterodimer) MMR complexes were present, was intermediate with hMutSα alone, and least with hMutSβ alone. Additionally, the effectiveness of adjuvant 5FU in patients with stage II/III CRC is dependent on a functional MMR system. We tested the hypothesis that stage II/III CRC patients whose cancers demonstrate EMAST (signifying hMSH3 and thus hMutSβ dysfunction) may not have the same response to adjuvant 5FU as EMAST-negative (fully functional MMR) CRC patients on survival. Methods: We analyzed 197 stage II/III sporadic colorectal cancers for which we had 5FU treatment and survival data. Archival DNA was extracted and analyzed for microsatellite instability (MSI: >2 of 5 NCI-recommended markers mutated) and EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). We generated Kaplan-Meier survival curves and utilized the log-rank test to determine statistical differences between groups. Results: We identified 52 (26%) MSI cancers and 93 (47%) EMAST-positive cancers, with 38 cancers showing both MSI and EMAST. Sixty-one patients (31%) received adjuvant 5FU chemotherapy, and median follow-up for all patients was 45 months. After excluding patients with MSI cancers that could confound the analysis, patients with EMAST-positive colon cancer did not improve survival with adjuvant 5FU (P>0.05) whereas patients with EMASTnegative colon cancer improved survival with adjuvant 5FU treatment (P=0.024). Including patients with MSI cancers did not alter the outcome for patients with EMAST-positive or EMAST-negative cancers treated with 5FU, and 5FU did not improve survival for patients with MSI cancers. Conclusions: There is improved survival in stage II/III CRC patients with EMAST-negative cancers after adjuvant 5FU-based chemotherapy that does not extend to patients with EMAST-positive cancers. The presence of hMSH3 (and thus hMutSβ function) is likely additive with hMutSα function within EMAST-negative tumors to improve the effectiveness of adjuvant 5FU for patient survival. Patients with MSI cancers do not improve their survival with adjuvant 5FU as previously reported. Determining the EMAST (and MSI) status of CRC patients may predict their response to 5FU-based chemotherapy. The presence of EMAST (and MSI) is one reason for the reduced effectiveness of adjuvant 5FU therapy in CRC patients.
S-690