Seon Ae Roh

Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells

Autophagy is a catabolic process involved in cell death and in cell protective mechanism. Autophagic cell death is differentiated from apoptosis by the presence of double or multiple-membrane enclosed vesicles, and the ATG proteins are essential for the formation of these autophagic vesicles. Here, we show that ATG6/Beclin-1 is a novel caspase substrate. ATG6 is directly cleaved by caspases in a process inhibited by the pan caspase inhibitor, zVAD. Ectopic expression of ATG6 suppresses cell death while reduction of ATG6 levels by siRNA sensitizes cells to TRAIL-induced cell death. Also, the inhibition of caspases leads to an increase in autophagy. These results suggest that caspase-mediated cleavage of ATG6 links the apoptotic and autophagic signaling pathways.

Novel Single-Nucleotide Polymorphism Markers Predictive of Pathologic Response to Preoperative Chemoradiation Therapy in Rectal Cancer Patients

PURPOSE Studies aimed at predicting individual responsiveness to preoperative chemoradiation therapy (CRT) are urgently needed, especially considering the risks associated with poorly responsive patients. METHODS AND MATERIALS A 3-step strategy for the determination of CRT sensitivity is proposed based on (1) the screening of a human genome-wide single-nucleotide polymorphism (SNP) array in correlation with histopathologic tumor regression grade (TRG); (2) clinical association analysis of 113 patients treated with preoperative CRT; and (3) a cell-based functional assay for biological validation. RESULTS Genome-wide screening identified 9 SNPs associated with preoperative CRT responses. Positive responses (TRG 1-3) were obtained more frequently in patients carrying the reference allele (C) of the SNP CORO2A rs1985859 than in those with the substitution allele (T) (P=.01). Downregulation of CORO2A was significantly associated with reduced early apoptosis by 27% (P=.048) and 39% (P=.023) in RKO and COLO320DM colorectal cancer cells, respectively, as determined by flow cytometry. Reduced radiosensitivity was confirmed by colony-forming assays in the 2 colorectal cancer cells (P=.034 and .015, respectively). The SNP FAM101A rs7955740 was not associated with radiosensitivity in the clinical association analysis. However, downregulation of FAM101A significantly reduced early apoptosis by 29% in RKO cells (P=.047), and it enhanced colony formation in RKO cells (P=.001) and COLO320DM cells (P=.002). CONCLUSION CRT-sensitive SNP markers were identified using a novel 3-step process. The candidate marker CORO2A rs1985859 and the putative marker FAM101A rs7955740 may be of value for the prediction of radiosensitivity to preoperative CRT, although further validation is needed in large cohorts.

Genome-wide identification of chemosensitive single nucleotide polymorphism markers in colorectal cancers.

Improved methods for predicting chemoresponsiveness involving the identification of polymorphic markers is highly desirable, considering narrow therapeutic index and frequent resistance to anti‐cancer regimens. The genome‐wide screening of chemosensitive single nucleotide polymorphisms (SNPs) was undertaken in association with in vitro chemosensitivity assays in 104 colorectal cancer patients for the initial screening step. Allele frequency, linkage disequilibrium, potential function, and Hardy–Weinberg equilibrium of the candidate SNPs were then determined for the identifying step. Finally, clinical association analysis in the other 260 evaluable patients or cell viability assays of transfected RKO cells was used to verify candidate SNPs for the validation step. In total, 12 SNPs to six regimens were initially chosen during the screening and identifying steps. In patients receiving fluoropyrimidine‐based adjuvant chemotherapy, the substitution alleles of GPC5 rs553717 (AA) correlated significantly with tumor recurrence and shorter disease‐free survival (P = 0.019 and 0.023, respectively). Interestingly, RKO cells expressing mutant GPC5 showed enhanced cell death in response to 5‐FU in cytotoxicity assays. Patients that were homozygous for the reference alleles SSTR4 rs2567608 (AA) and EPHA7 rs2278107 (TT) showed lower disease control rates in response to irinotecan and oxaliplatin regimens, respectively, than those with substitution alleles (P = 0.022 and 0.014, respectively). Thus, we identified chemosensitive SNP markers using a novel three step process of genome‐wide analysis consisting of in vitro screening, identification, and validation. The candidate chemosensitive SNP markers identified in our study, including those identified in vitro, can now be further verified in a large cohort study.

Poorly Differentiated Colorectal Cancers

OBJECTIVES To evaluate the association of microsatellite instability (MSI) with clinicopathologic features and oncologic outcomes in patients with poorly differentiated colorectal cancer (PD). METHODS Study patients were divided into well-differentiated colorectal cancer (WD) and PD, which were compared according to histologic differentiation and MSI status. RESULTS Among 1,941 patients, PD was more frequent among microsatellite-unstable tumors (23.6%) than among microsatellite-stable (MSS) tumors (4.2%, P < .001). Patients with PD had worse 4-year overall survival rates than patients with WD (78.6% vs 88.2%, P = 0.010). Compared with MSS-PD tumors, MSI-PD tumors were characterized by right-colon predilection, larger size, and infrequent lymph node metastasis (P < .001 to P = .007). CONCLUSIONS The clinicopathologic characteristics of PD were closely associated with those of MSI. The outcomes of MSI-PD tumors were better than those of MSS-PD tumors, but this finding did not reach statistical significance.

Polypyrimidine tract-binding protein 1-mediated down-regulation of ATG10 facilitates metastasis of colorectal cancer cells.

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells.

Feasibility of proposed single-nucleotide polymorphisms as predictive markers for targeted regimens in metastatic colorectal cancer.

Background:Surrogate biomarkers for metastatic colorectal cancer (mCRC) are urgently needed to achieve the best outcomes for targeted therapy.Methods:A clinical association analysis was performed to examine the three single-nucleotide polymorphisms (SNPs) that were previously proposed as markers of chemosensitivity to the cetuximab (124 patients) and bevacizumab regimens (100 patients) in mCRC patients. In addition, biological correlations were examined for the candidate SNPs in terms of their regulatory pathway.Results:For cetuximab regimens, patients homozygous for the wild-type alleles (GG) of LIFR rs3729740 exhibited a 1.9 times greater overall response rate (ORR) and 1.4 months longer progression-free survival (PFS) than those homozygous or heterozygous for the mutant allele (GA and AA; P=0.022 and 0.027, respectively). For bevacizumab regimens, patients homozygous for the minor alleles (TT) of ANXA11 rs1049550 exhibited an ORR twice as high as those homozygous or heterozygous for the ancestral allele (CC and CT; P=0.031). Overall response rate gain was achieved up to 10% in patients with wild-type LIFR rs3729740 patients either with wild-type KRAS or skin toxicity (P=0.001) respectively. Specifically in clones treated with cetuximab and bevacizumab regimens, active p-ERK and MMP-9 expressions were significantly reduced in clones expressing wild-type LIFR rs3729740 (P=0.044) and in those expressing minor-type ANXA11 rs1049550 (P=0.007), respectively.Conclusion:LIFR rs3729740 and possibly ANXA11 rs1049550 may be useful as biomarkers for predicting whether mCRC patients are sensitive to relevant target regimens, although further validation in large cohorts is needed.

Complex Behavior of ALDH1A1 and IGFBP1 in Liver Metastasis from a Colorectal Cancer

Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, ALDH1A1 and IGFBP1 were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001–0.003) and suppressed invasiveness by ≥3-fold over control cells (p < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (p < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on in vitro cell assays, persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/IGFBP1-transfected mice vs. SW480/vector- and SW480/ALDH1A1-transfected mice, 4/8 vs. 0/10, p = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.

Epigenetic regulation of KLHL34 predictive of pathologic response to preoperative chemoradiation therapy in rectal cancer patients.

PURPOSE Prediction of individual responsiveness to preoperative chemoradiation therapy (CRT) is urgently needed in patients with poorly responsive locally advanced rectal cancer (LARC). METHODS AND MATERIALS Candidate methylation genes associated with radiosensitivity were identified using a 3-step process. In the first step, genome-wide screening of methylation genes was performed in correlation with histopathologic tumor regression grade in 45 patients with LARC. In the second step, the methylation status of selected sites was analyzed by pyrosequencing in 67 LARC patients, including 24 patients analyzed in the first step. Finally, colorectal cancer cell clones with stable KLHL34 knockdown were generated and tested for cellular sensitivity to radiation. RESULTS Genome-wide screening identified 7 hypermethylated CpG sites (DZIP1 cg24107021, DZIP1 cg26886381, ZEB1 cg04430381, DKK3 cg041006961, STL cg00991794, KLHL34 cg01828474, and ARHGAP6 cg07828380) associated with preoperative CRT responses. Radiosensitivity in patients with hypermethylated KLHL34 cg14232291 was confirmed by pyrosequencing in additional cohorts. Knockdown of KLHL34 significantly reduced colony formation (KLHL34 sh#1: 20.1%, P=.0001 and KLHL34 sh#2: 15.8%, P=.0002), increased the cytotoxicity (KLHL34 sh#1: 14.8%, P=.019 and KLHL34 sh#2: 17.9%, P=.007) in LoVo cells, and increased radiation-induced caspase-3 activity and the sub-G1 population of cells. CONCLUSIONS The methylation status of KLHL34 cg14232291 may be a predictive candidate of sensitivity to preoperative CRT, although further validation is needed in large cohorts using various cell types.

The prognostic significance and treatment modality for elevated pre- and postoperative serum CEA in colorectal cancer patients

Purpose The purpose of this study was to evaluate the prognostic significance of serum CEA (s-CEA) changes in colorectal cancer (CRC) patients with sustained elevated postoperative s-CEA levels. Methods Between January 1999 and December 2008, 9,380 CRC patients underwent surgery. Curative resection was performed in 1,242 CRC patients with high preoperative s-CEA levels (>6 ng/mL). High s-CEA levels were normalized in 924 patients (74.4%) within 2 weeks from surgery, whereas high s-CEA levels were persistent in 318 patients (25.6%). Patients were divided into 2 groups according to their postoperative s-CEA levels: group 1 (37 patients with a 1-year postoperative s-CEA>6 ng/mL) and group 2 (281 patients with a 1-year postoperative s-CEA≤6 ng/mL). Results A postoperative recurrence was identified in 24 patients (64.9%) in group 1 and 65 patients (23.1%) in group 2 (P < 0.001). A curative resection after recurrence was performed in 22 patients (33.8%) from group 2, but no patients from group 1 (P = 0.001). The 5-year overall survival and time to recurrence were significantly lower in patients with recurrent cancer in group 1 (P < 0.001). Conclusion Patients with persistent elevated postoperative s-CEA levels are at high risk for recurrence and a low survival rate. More intensive surveillance of patients with high postoperative s-CEA levels should be mandatory.

Abstract 4992: Zkscan3 facilitates invasion of colorectal cancer associated with ceacam5

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: ZKSCAN3 is over-expressed in invasive colonic tumor cells and regulate the expression of several genes favoring tumor progression including integrin β4. We evaluated the role of ZKSCAN3 in invasive signaling pathway with stage 4 colorectal cancer (CRC). Materials and methods: 119 metastatic CRC (mCRC) patients who palliatively or curatively resected and 331 sporadic CRC patients who curatively resected from January 2004 to December 2009 were used to identify correlations between single-nucleotide polymorphism (SNP) rs733743 and clinically prognostic parameters. To validation of prior correlation, the genotypes of SNP identified by pyrosequencing using 81 patients (33 with mCRC and 48 CRC patients) and CRC cell lines. We then performed immunohistochemistry (IHC) and Western blot on 36 mCRC patients and 49 CRC patients. Up-regulated putative down-stream targets included genes contributing tumor aggressiveness and invasiveness; VEGF, cyclin D2, Akt, phospho-Akt, integrin β4, KRAS, CEACAM5, and integrin α5β1. To test biological utility assay, ZKSCAN3 mRNA was knocked down by ZKSCAN3 specific siRNA in metastatic CRC cells (LOVO) and ZKSCAN3 cDNA was overexpressed in the low-ZKSCAN3 expressed CRC cells (HCT116). Results: Wild-type alleles (GG) of ZKSCAN3 rs733743 was related with male dominant, family history of malignancy, high CEA concentration, and mCRC. The wild-type alleles of ZKSCAN3 rs733743 using tissue sample was related with lymphvascular invasion. ZKSCAN3 IHC positive tissue was related with lymphovascular invasion. ZKSCAN expression was higher in LOVO and SW620 cell lines which were derived from metastatic site. ZKSCAN3 high groups were related with CEA, integrin β4, VEGF, and AKT and ZKSCAN3 low groups were related with RAS, VEGF, AKT, and PAKT in metastatic tumor tissue. Therefore, ZKSCAN3 seemed to be related with CEA and integrin β4 to metastasis. Additional CEA expression was observed a significant decrease in ZKSCAN3 knockdowned LOVO cell (1.0 vs 0.89, P= 0.046) and invasiveness of the ZKSCAN3 overexpressed HCT116 cells was higher in CEA coated filters (75.6±6.0 vs 91.3±4.5, P=0.023). Conclusion: ZKSCAN3 is related with colorectal tumor progression and invasion. ZKSCAN3 overexpression tumor may facilitate metastasis of colorectal cancer associated with CEACAM5. Citation Format: Seon Ae Roh, Chan Wook Kim, Ka Hee Tak, Jin Cheon Kim. Zkscan3 facilitates invasion of colorectal cancer associated with ceacam5. [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 4992. doi:10.1158/1538-7445.AM2014-4992