- Email: [email protected]
H+ Transporter Protein With a Promising Potential As a Prognostic Biomarker in Colorectal Cancer
knockdown experiments with TMCO3 (Silencer Select siRNA, Life Technologies). The successful induction of siRNA against TMCO3 was identified by Taqman RT-PCR and western immunoblotting. Cell proliferation ability was analyzed using MTT assay. Cell invasion and migration status was measured with Matrigel Invasion Chambers. Induction of apoptosis was analyzed by Annexin V & Dead Cell Kit (Millipore). In addition, the transcript levels of TMCO3 were analyzed in matched pairs of tumor and normal mucosa tissues from a cohort of 122 CRC patients. All patients were categorized into TMCO3-Upregulated and TMCO3-Downregulated groups, and clinicopathological features were analyzed. Results: The expression levels of TMCO3 were significantly reduced in si-TMCO3 cells, compared to that of control siRNA cells, in both SW480 and CACO2 cell lines. TMCO3-knockdown resulted in decreased cellular proliferation, invasion and migration, but enhanced apoptosis, compared to control cells. In CRC specimens, TMCO3 expression increased in a stepwise manner in stage I-IV tumors, and CRCs with up-regulated expression of TMCO3 demonstrated significantly poor overall and disease-free survival compared to the down-regulated group. Conclusion: Our results provide first evidence for the role of TMCO3 in CRC, and illustrate its clinical usefulness as a potential prognostic biomarker in this malignancy. Our findings provide a compelling rationale for TMCO3 as an indispensable protein in CRC progression, and expound upon its value as a promising candidate for targeted therapy.
Mitochondrial Alterations in Early Colon Carcinogenesis: Evidence for Warburg Effect in the Initiation of Colorectal Cancer (CRC) Carcinogenesis Mart DeLaCruz, Bilal Latif, Anuj Chhaparia, Navneet Momi, Ramesh K. Wali, Hemant K. Roy Background: There is emerging realization of the central role of metabolism in carcinogenesis with the Warburg effect (preferential utilization of glycolysis in normoxic environment) (e.g. Weinberg, Cell 2012). We have previously noted an increase in microvascular blood supply in the predysplastic colonic mucosa (field carcinogenesis) (Gut 2005, Gastro 2008, Clin Cancer Res 2009, Cancer Prev Res 2010) which may reflect altered metabolism, however molecular determinants remain unexplored. The role of mitochondria in CRC is well established in frank tumors with dysfunction reflected by increased fission leading to less efficient oxidative phosphorylation, enabling shunting of precursors for cell growth. However, the presence of mitochondrial dysfunction and Warburg effect in early colon carcinogenesis has yet been explored. Therefore, we chose to investigate potential mitochondrial changes in the pre-malignant colon. Methods: To investigate mitochondrial changes we incorporated both human and animal studies: For human studies we obtained biopsies from endoscopically normal rectal muscosa from 80 patients undergoing screening colonoscopies. Samples were processed for gDNA and RNA isolation. Real Time PCR was conducted to assess mitochondrial mass, OPA-1, UCP2, and PKM2 expression. For animal experiments we utilized the Polypopsis in Rat Colon (Pirc) rat, which possesses an APC truncation, consistent with the initiating factor in ~80% of sporadic CRCs. 7 Pirc rats with age-matched controls were euthanized after 24 wks of age. Colons were excised from these animals to obtain colonic gDNA and RNA. Real Time PCR was conducted to assess mitochondrial mass, OPA-1 and UCP2 expression. Results: In patient biopsies, mitochondrial mass was shown to be upregulated with adenomal presence (~72% increase, p=0.05). Pirc rats were also shown to possess a 63.6% induction of mitochondrial mass (p=0.03). OPA-1 expression was upregulated in both patients with adenomas as well as Pirc rats (49%, p=0.05 and 67%, p<0.03 respectively). Induction of UCP2 was detected in patients with adenomas (99%, p<0.04) and in Pircs (101%, p=0.05). Interestingly, patients with lesions had a 80% increase in PKM2 (p=0.002), suggestive of a Warburg effect in early colon carcinogenesis. Conclusions: We demonstrate for the first time, that in the predysplastic mucosa there is profound metabolic alterations consistent with a Warburg effect. Specifically, we noted increased mitochondria mass (fission) which suppresses oxidative phosphorylation. Aside from structural alterations, electron transport system is further inhibited by overexpression of UCP2. Biologically, this provides insight into early metabolic events in colon carcinogenesis. Clinically, these insights may enable novel biomarkers for early detection and also a myriad of potentially "druggable" targets for chemoprevention.
Sa1913 The Effect of Prediagnostic Aspirin Use on the Prognosis of Stage III Colorectal Cancer Bun Kim, Soo Jung Park, Sung Pil Hong, Jae Hee Cheon, Won Ho Kim, Tae Il Kim Background: Many studies have suggested that the regular use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, has a protective effect and survival benefit on colorectal cancer (CRC). However, recent data suggest that CRCs have different responses to NSAIDs depending on the timing of NSAID initiation, duration of NSAID use, and molecular characteristics of the tumor. We hypothesized that tumors that developed despite long-term exposure to aspirin might induce different tumor characteristics such as poor response to adjuvant chemotherapy and more recurrence. However, there has been little data about prognosis in prediagnostic aspirin users comparing to aspirin non-users, particularly in stage III CRC. Therefore, we investigated the effect of long-term prediagnostic aspirinuse on tumor recurrence in stage III CRC and showed it in an experimental model. Methods: From 2007 to 2009, patients who were diagnosed with stage III CRC were recruited, and their medical records were retrospectively analyzed. Patients were divided into prediagnostic aspirin users (who used aspirin for more than three months continuously before CRC diagnosis) and non-users (who did not use of aspirin and NSAIDs). The two groups were compared in terms of recurrence, cancer-specific mortality, disease-free survival (DFS), and cancer-specific survival. In an experimental study, three CRC cell lines (Caco2, SW480, and DLD-1) were pretreated with aspirin (1 mM) for eight days or 28 days to make aspirinresistant cells, treated with 5-fluorouracil (5-FU; 2 μM), and apoptosis was measured with flow cytometry using Annexin-V and propidium iodide double staining. Results: Compared with the aspirin non-users (N=565), the prediagnostic aspirin users (N=121) were not different in terms of baseline characteristics including tumor characteristics, except for comorbidities and diabetes medication and statin use, which were higher in the prediagnostic aspirin users. Recurrence and cancer-specific mortality in stage III CRC were significantly higher in prediagnostic aspirin users than non-users (46.7% vs. 32.3%, p=0.003 and 32.2% vs. 19.8%, p=0.003, respectively). Survival analysis using Cox proportional hazards modeling demonstrated that DFS was significantly worse in prediagnostic aspirin users than non-users (HR, 1.525 (1.018-2.286); p=0.041). In cell line experiments, long-term aspirin pretreatment induced an increase in 5-FU-induced apoptosis in SW480 cells compared with control treatment without aspirin pretreatment. However, Caco2 cells showed a significant decrease of apoptosis in the same experiments and no change in DLD1 cells. Conclusion: Prediagnostic long-term aspirin use in stage III CRC could be a negative prognostic factor depending on the characteristics of the CRC, suggesting the development of different tumor characteristics of aspirin-resistant CRC.
Figure 1. Mitochondrial changes with adenomal presence in humans as well as Pirc rat.
Sa1914 The Genomic Landscape of Human Colon Adenomas Reveals Early Driver Mutations and a TGF-β-CEA Regulated Profile Vipin K. Menon, Gottumukkala S. Raju, Jian Chen, Xiaoping Su, Avijit Majumdar, JiHyun Shin, Shulin Li, Kirti Shetty, Xifeng Wu, Brian R. Weston, Ethan Miller, John R. Stroehlein, Marta L. Davila, Mehnaz A. Shafi, Asif Rashid, Bhaskar V. Kallakury, Selvi Thirumurthi, John S. McMurray, Sue-Hwa Lin, Wilma Jogunoori, Lopa Mishra Objective: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world with 143,700 newly diagnosed cases in 2012. One of the contributing factors that have newly emerged is the rapid development of cancers in small, sessile adenomas which may have been overlooked. Despite many advances in cancer genetics, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support identification and effective preventive measures for high risk adenomas leading to CRC. We hypothesized that identifying high risk patients through genomic analysis of adenomas could potentially lead to an early intervention therapy. Methods: Whole Genome Sequence (WGS) and Whole Exome Sequence analyses were performed for 4 pairs of normal controls (two from proximal and two from the distal colons) and colorectal adenomas (2 sessile serrated (SSA) and 2 tubulovillous adenomas (TVA) less than 1 cm in size). Further transcriptome sequence analysis was performed in seven pairs of control-test matched adenomas (6 TVA and 1 SSA). Results:1. WGS revealed a hyper-mutator profile in two of the samples (1 SSA and 1 TVA) with 1709 mutations after normalization with normal paired samples, and an average mutation frequency of 0.55 mutations per 106 bases. 2. Seven of the eleven adenomas had aberrant mutational profiles. 3. We observed distinct mutational signatures among the samples, two with high, two intermediate and three low mutational rates. 4. Wnt and p53 pathways were altered in 6 out of 11 adenomas (1 SSA and 4TVAs). 5. In 37% of samples we observed C:T>G:A transitional single nucleotide substitutions within the mutations spectrum. 6. Subtle localized hyper mutation (kataegis) was observed among two of the samples when inter-mutation distance of the samples was
Figure 2. UCP2 expression with adenomal presence in humans as well as Pirc rat. Sa1912 TMCO3 - A Novel Na+/H+ Transporter Protein With a Promising Potential As a Prognostic Biomarker in Colorectal Cancer Kunitoshi Shigeyasu, Yoshinaga Okugawa, Shusuke Toden, Ajay Goel Background: As per Warburg effect, the pH in the cancerous regions is significantly lower than the corresponding normal area. Dysregulation of pH affects tumor growth, cell viability, proliferation and motility. Cancer cells up-regulate acid transportation proteins to maintain intra-cellular pH homeostasis. TMCO family has been suggested to function as a potential regulator for Na+/H+ levels in the cells, but the basic molecular mechanisms and specific proteins within this family that regulate intracellular pH remain poorly understood. Aim: In this study, we investigated the role of TMCO3 as a potential Na+/H+ transporter within cancer cells, and the functional role for this protein in promoting colorectal cancer (CRC). Methods: We used two human CRC cell lines, SW480 and CACO2, and performed siRNA
S-353
AGA Abstracts
AGA Abstracts
Sa1911
Mitochondrial Alterations in Early Colon Carcinogenesis: Evidence for Warburg Effect in the Initiation of Colorectal Cancer (CRC) Carcinogenesis Mart DeLaCruz, Bilal Latif, Anuj Chhaparia, Navneet Momi, Ramesh K. Wali, Hemant K. Roy Background: There is emerging realization of the central role of metabolism in carcinogenesis with the Warburg effect (preferential utilization of glycolysis in normoxic environment) (e.g. Weinberg, Cell 2012). We have previously noted an increase in microvascular blood supply in the predysplastic colonic mucosa (field carcinogenesis) (Gut 2005, Gastro 2008, Clin Cancer Res 2009, Cancer Prev Res 2010) which may reflect altered metabolism, however molecular determinants remain unexplored. The role of mitochondria in CRC is well established in frank tumors with dysfunction reflected by increased fission leading to less efficient oxidative phosphorylation, enabling shunting of precursors for cell growth. However, the presence of mitochondrial dysfunction and Warburg effect in early colon carcinogenesis has yet been explored. Therefore, we chose to investigate potential mitochondrial changes in the pre-malignant colon. Methods: To investigate mitochondrial changes we incorporated both human and animal studies: For human studies we obtained biopsies from endoscopically normal rectal muscosa from 80 patients undergoing screening colonoscopies. Samples were processed for gDNA and RNA isolation. Real Time PCR was conducted to assess mitochondrial mass, OPA-1, UCP2, and PKM2 expression. For animal experiments we utilized the Polypopsis in Rat Colon (Pirc) rat, which possesses an APC truncation, consistent with the initiating factor in ~80% of sporadic CRCs. 7 Pirc rats with age-matched controls were euthanized after 24 wks of age. Colons were excised from these animals to obtain colonic gDNA and RNA. Real Time PCR was conducted to assess mitochondrial mass, OPA-1 and UCP2 expression. Results: In patient biopsies, mitochondrial mass was shown to be upregulated with adenomal presence (~72% increase, p=0.05). Pirc rats were also shown to possess a 63.6% induction of mitochondrial mass (p=0.03). OPA-1 expression was upregulated in both patients with adenomas as well as Pirc rats (49%, p=0.05 and 67%, p<0.03 respectively). Induction of UCP2 was detected in patients with adenomas (99%, p<0.04) and in Pircs (101%, p=0.05). Interestingly, patients with lesions had a 80% increase in PKM2 (p=0.002), suggestive of a Warburg effect in early colon carcinogenesis. Conclusions: We demonstrate for the first time, that in the predysplastic mucosa there is profound metabolic alterations consistent with a Warburg effect. Specifically, we noted increased mitochondria mass (fission) which suppresses oxidative phosphorylation. Aside from structural alterations, electron transport system is further inhibited by overexpression of UCP2. Biologically, this provides insight into early metabolic events in colon carcinogenesis. Clinically, these insights may enable novel biomarkers for early detection and also a myriad of potentially "druggable" targets for chemoprevention.
Sa1913 The Effect of Prediagnostic Aspirin Use on the Prognosis of Stage III Colorectal Cancer Bun Kim, Soo Jung Park, Sung Pil Hong, Jae Hee Cheon, Won Ho Kim, Tae Il Kim Background: Many studies have suggested that the regular use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, has a protective effect and survival benefit on colorectal cancer (CRC). However, recent data suggest that CRCs have different responses to NSAIDs depending on the timing of NSAID initiation, duration of NSAID use, and molecular characteristics of the tumor. We hypothesized that tumors that developed despite long-term exposure to aspirin might induce different tumor characteristics such as poor response to adjuvant chemotherapy and more recurrence. However, there has been little data about prognosis in prediagnostic aspirin users comparing to aspirin non-users, particularly in stage III CRC. Therefore, we investigated the effect of long-term prediagnostic aspirinuse on tumor recurrence in stage III CRC and showed it in an experimental model. Methods: From 2007 to 2009, patients who were diagnosed with stage III CRC were recruited, and their medical records were retrospectively analyzed. Patients were divided into prediagnostic aspirin users (who used aspirin for more than three months continuously before CRC diagnosis) and non-users (who did not use of aspirin and NSAIDs). The two groups were compared in terms of recurrence, cancer-specific mortality, disease-free survival (DFS), and cancer-specific survival. In an experimental study, three CRC cell lines (Caco2, SW480, and DLD-1) were pretreated with aspirin (1 mM) for eight days or 28 days to make aspirinresistant cells, treated with 5-fluorouracil (5-FU; 2 μM), and apoptosis was measured with flow cytometry using Annexin-V and propidium iodide double staining. Results: Compared with the aspirin non-users (N=565), the prediagnostic aspirin users (N=121) were not different in terms of baseline characteristics including tumor characteristics, except for comorbidities and diabetes medication and statin use, which were higher in the prediagnostic aspirin users. Recurrence and cancer-specific mortality in stage III CRC were significantly higher in prediagnostic aspirin users than non-users (46.7% vs. 32.3%, p=0.003 and 32.2% vs. 19.8%, p=0.003, respectively). Survival analysis using Cox proportional hazards modeling demonstrated that DFS was significantly worse in prediagnostic aspirin users than non-users (HR, 1.525 (1.018-2.286); p=0.041). In cell line experiments, long-term aspirin pretreatment induced an increase in 5-FU-induced apoptosis in SW480 cells compared with control treatment without aspirin pretreatment. However, Caco2 cells showed a significant decrease of apoptosis in the same experiments and no change in DLD1 cells. Conclusion: Prediagnostic long-term aspirin use in stage III CRC could be a negative prognostic factor depending on the characteristics of the CRC, suggesting the development of different tumor characteristics of aspirin-resistant CRC.
Figure 1. Mitochondrial changes with adenomal presence in humans as well as Pirc rat.
Sa1914 The Genomic Landscape of Human Colon Adenomas Reveals Early Driver Mutations and a TGF-β-CEA Regulated Profile Vipin K. Menon, Gottumukkala S. Raju, Jian Chen, Xiaoping Su, Avijit Majumdar, JiHyun Shin, Shulin Li, Kirti Shetty, Xifeng Wu, Brian R. Weston, Ethan Miller, John R. Stroehlein, Marta L. Davila, Mehnaz A. Shafi, Asif Rashid, Bhaskar V. Kallakury, Selvi Thirumurthi, John S. McMurray, Sue-Hwa Lin, Wilma Jogunoori, Lopa Mishra Objective: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world with 143,700 newly diagnosed cases in 2012. One of the contributing factors that have newly emerged is the rapid development of cancers in small, sessile adenomas which may have been overlooked. Despite many advances in cancer genetics, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support identification and effective preventive measures for high risk adenomas leading to CRC. We hypothesized that identifying high risk patients through genomic analysis of adenomas could potentially lead to an early intervention therapy. Methods: Whole Genome Sequence (WGS) and Whole Exome Sequence analyses were performed for 4 pairs of normal controls (two from proximal and two from the distal colons) and colorectal adenomas (2 sessile serrated (SSA) and 2 tubulovillous adenomas (TVA) less than 1 cm in size). Further transcriptome sequence analysis was performed in seven pairs of control-test matched adenomas (6 TVA and 1 SSA). Results:1. WGS revealed a hyper-mutator profile in two of the samples (1 SSA and 1 TVA) with 1709 mutations after normalization with normal paired samples, and an average mutation frequency of 0.55 mutations per 106 bases. 2. Seven of the eleven adenomas had aberrant mutational profiles. 3. We observed distinct mutational signatures among the samples, two with high, two intermediate and three low mutational rates. 4. Wnt and p53 pathways were altered in 6 out of 11 adenomas (1 SSA and 4TVAs). 5. In 37% of samples we observed C:T>G:A transitional single nucleotide substitutions within the mutations spectrum. 6. Subtle localized hyper mutation (kataegis) was observed among two of the samples when inter-mutation distance of the samples was
Figure 2. UCP2 expression with adenomal presence in humans as well as Pirc rat. Sa1912 TMCO3 - A Novel Na+/H+ Transporter Protein With a Promising Potential As a Prognostic Biomarker in Colorectal Cancer Kunitoshi Shigeyasu, Yoshinaga Okugawa, Shusuke Toden, Ajay Goel Background: As per Warburg effect, the pH in the cancerous regions is significantly lower than the corresponding normal area. Dysregulation of pH affects tumor growth, cell viability, proliferation and motility. Cancer cells up-regulate acid transportation proteins to maintain intra-cellular pH homeostasis. TMCO family has been suggested to function as a potential regulator for Na+/H+ levels in the cells, but the basic molecular mechanisms and specific proteins within this family that regulate intracellular pH remain poorly understood. Aim: In this study, we investigated the role of TMCO3 as a potential Na+/H+ transporter within cancer cells, and the functional role for this protein in promoting colorectal cancer (CRC). Methods: We used two human CRC cell lines, SW480 and CACO2, and performed siRNA
S-353
AGA Abstracts
AGA Abstracts
Sa1911