EGFR-inhibitor (Cetuximab) is one of the main targeted drugs used for metastatic colorectal carcinoma (CRC). The benefit from Cetuximab appears to be limited to a subtype of patients, not for the patients with tumors harboring mutated BRAF or KRAS genes; unfortunately, it accounts for ~40-50% of CRC cases. Previous studies have connected higher expression levels of miR-378 to be commonly presented in patients without BRAF or KRAS mutants than in mutated CRCs. The microRNA-378 (miR-378) is coexpressed with PGC-1β and can be easily induced by fatty acid, for example lauric acid. Therefore, we hypothesized that elevation of miR-378 expression in mutated CRCs may stimulate the cell response to Cetuximab. Herein, seven CRC cell lines with confirmed mutation status were involved in two parallel experiments; directly in vitro transfected miR-378 mimics, and using lauric acid to indirectly induce the level of miR-378 in cells. After the increase of miR-378 in cells by either direct or indirect approaches, sensitivity to Cetuximab was restored in all BRAF mutants (p-value <0.0001-0.0003), and half of KRAS mutants CRC (p-value 0.039-0.007). Further evidence was gained by decreasing expression of MEK and ERK2 proteins after transfection with miR-378; it was similar to the indirect induction by lauric acid approach. In conclusion, the present study demonstrated that lauric acid may efficiently induce miR-378 expression in CRC mutants, and both BRAF and a subtype of KRAS mutants presented significantly improved sensitivity to Cetuximab. Notably, BRAF mutants could even be inhibited in cell proliferation after elevated concentration of miR-378 in cells without combining with targeted therapy. This new approach may shed new light on BRAF or KRAS mutation in CRC patients for clinical trial, since lauric acid may easily be obtain from natural food, and it is supposed to be harmless to the cardiovascular system.

The PI3K/Akt signaling pathway serves an essential role in various cellular processes, including cell growth, survival, cell motility, angiogenesis and cell metabolism. Loss of PTEN expression and hyperactivation of Akt can result in tumorigenesis. Previous studies observed expression of the Akt protein and absence of the PTEN protein in bladder cancer and non-small cell lung carcinoma tissues. The aim of the present study was to evaluate the expression status and prognostic value of PTEN and the PI3K/Akt signaling pathway in Taiwanese patients with upper tract urothelial carcinoma (UTUC). Archival formalin-fixed, paraffin-embedded (FFPE) tissues from 65 UTUC cases were stained via immunohistochemistry for PTEN, phosphorylated (p)Akt serine (Ser) 473 and pAkt threonine (Thr) 308 . The expression levels of each protein were significantly correlated with clinicopathological parameters. PTEN, pAkt Ser473 and pAkt Thr308 protein expression levels were higher in adjacent normal tissues compared with those in tumor tissues. Cytoplasmic PTEN protein expression levels were lower in high-stage tumors compared with those in low-stage tumors, and nuclear and cytoplasmic pAkt Thr308 protein expression levels were higher in high-grade tumors compared with those in low-grade tumors. Univariate analysis showed that high pathological tumor stage (pT2-4) [P=0.01; hazard ratio (HR)=3.40; 95% confidence interval (CI), 1.34–8.60], metastatic status (P=0.003; HR=3.55, 95% CI, 1.55–8.11), low cytoplasmic PTEN protein expression levels (P=0.016; HR=3.14; 95% CI, 1.24–7.95) and high cytoplasmic pAkt Ser473 protein expression levels (P=0.019, HR=2.71, 95% CI, 1.18–6.21) were predictive of poor overall survival. However, only metastatic status (P=0.031; HR=2.73; 95% CI, 1.10–6.78), low cytoplasmic PTEN protein expression levels (P=0.017; HR=3.29; 95% CI, 1.24–8.73) and high cytoplasmic pAkt Ser473 protein expression levels (P=0.027; HR=2.64; 95% CI, 1.12–6.23) remained significant in the multivariate analysis. Kaplan-Meier survival analysis showed that high T stage, metastasis, low expression levels of cytoplasmic PTEN protein and high expression levels of cytoplasmic pAkt Ser473 protein were significantly associated with poor survival (P=0.006, 0.001, 0.011 and 0.014, respectively). Co-expression of PTEN low /pAkt Ser473/high and pAkt Thr308/high phenotypes was associated with a less favorable overall survival (P=0.001). Overall, the present findings demonstrated that low expression levels of PTEN and high expression levels of pAkt Ser473 and pAkt Thr308 were predictors for poor overall survival in patients with UTUC.

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer, more then 40% of patients with CRC have KRAS mutations are resistant to anti-EGFR therapy. Lower expression of microRNA-378 (miR-378) was reported to be associated with CRC contain with KRAS or BRAF mutation when compared with wild type of CRC cells. As known that miR-378 is derived from the 3′-UTR of PGC-1β (Peroxisome proliferator activated receptor gamma coactivator-1β) gene, and expressed abundantly could be stimulated by lauric acid. Therefore, we hypothesized once increase the expression level of miR-378 might restore the sensitivity to anti-EGFR sensitizing of cetuximab in KRAS or BRAF mutated colon cancer cells. Herein, we firstly enhanced expression level of miR-378 in mutants via transfection method, and further treated with cetuximab, hopefully, the mutants re-sensitizing to previously failed therapies could be demonstrated. Seven colon cancer cell lines with confirmed KRAS and BRAF statuses were used in current study. Untreated original cells, cells treated with cetuximab, cells transfected with miR-378, and miR-378 transfected cells combined with cetuximab treatment were included. Cell viabilities were then measured for all, and compared to each other group. Our results showed, after transfected miR-378, although most all of KRAS mutants increased cell viabilities except the BRAF mutated CRC cells; nevertheless, the mutants significantly responsed to cetuximab treatment could be observed when compared to the cells treated with cetuximab only. The results indicated that increased the level of miR-378 combined anti-EGFR drug treatment could lead cell viabilities significantly decreased in either BRAF mutants or 50% of KRAS mutated cells. Obviously, re-sensitized to anti-EGFR drug was coming off following transfected with miR-378 in mutated cells. Based on above findings, we then further tried to use lauric acid (C12:0) to enhance the expression level of miR-378 in the cancer cells, and hopefully it would benefit to those CRC cases who ever failed to anti-EGFR antibody treatment. The similar results showed once enhancing the expression level of miR-378, and then treated with anti-EGFR antibody, consequentially lower cell survival could be observed. Lower protein expression of ERK 1/2 has been also been observed in lauric acid treated CRC cells. All the results tend to disclose lauric acid might be an element, which could trigger KRAS cells restored sensitivity to anti-EGFR based drug. In conclusion, elevating miR-378 expression level in mutated CRC cells by lauric acid, which could be easily derived from olive oil, would allow drug re-sensitization in both BRAF and 50% of KRAS mutated cells, the cells were all significantly suppressed by cetuximab. The role of miR-378 in modulating anti-EGFR sensitivity is obvious; therefore, it was strongly suggested to be a potentially new strategy for the clinical CRC patients who have failed anti-EGFR drug treatment. Note: This abstract was not presented at the meeting. Citation Format: Shih-Chun Bian, Wai-Hung Leung, Yeu-Jye Pang, Yu-Wen Wu, Jing-Jung Chen, Wen-Hui Weng. Increasing microRNA-378 to enhance sensitivity of EGFR inhibitor in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3984. doi:10.1158/1538-7445.AM2015-3984

Cholangiocarcinoma (CCA) is an adenocarcinoma of the biliary tract, which is considered to be an incurable and rapidly lethal disease. A major problem with CCA surveillance is the lack of reliable biomarkers, therefore, still less no significant aspects of client-centered therapy in clinical treatment so far. Here, we established a 25th week thioacetamide (TAA)-induced cholangiocarcinoma rat cell line model named Chang Gung CCA (CGCCA), aim to figure out the early alterations of cytogenetics, molecular and protein expressed characteristics of CCA. Array comparative genomic hybridization (aCGH) and Spectral karyotyping (SKY), gene expression microarray and immunohistochemistry were performed on either rat CCA tissues or CGCCA cell line in this study. Our results showed complicated alterations of genetic gain or loss on different chromosomal regions could be observed. The data compared with the gene expression profile, thirty-three genes aberrations could be further identified; for example, tumor metastasis-related genes ANXA1 and CLCA3, the genes MMP7 and MMP12 are related with tumor invasion, and proliferation-related genes PKM2 and OSMR have been pinpointed; The number of chromosome ranging between 50 to 56 diplody (2n); despite the apparently high incidence of chromosomal translocation was revealed by cytogenetic analyses, ring and/or giant rod marker chromosomes were detected in almost every CGCCA cells, and the genetic materials are mainly included chromosomes 4 and 8. Herein, we concluded ANXA1, CLCA3, MMP7, MMP12, PKM2 and OSMR genes could be considered as potential biomarkers for the risk of disease. Supernumerary ring or giant rod marker chromosomes could also be considered as a characteristic may be involved in the late stage of CCA. However, the genes involved in the tumorigenesis should be further investigated the function of the genes. In order to verify the clinical significance and potentially to be as therapeutic targets in the future, to confirm onto a series of human CCA tissues are necessary. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3881. doi:10.1158/1538-7445.AM2011-3881

It is known that epidermal growth factor receptor (EGFR) inhibitor, cetuximab, does not respond to cells with KRAS or BRAF mutations; therefore, a large proportion (more than 40%) of colorectal cancer patients are unable to receive this type of target therapy, and can only undergo surgical as a way of management. In this study, we aim to provide an alternative therapeutic strategy to KRAS or BRAF mutated neoplasms. Our previous studies have demonstrated that there may be better sensitivity to anti-EGFR antibody therapy in KRAS or BRAF mutants with increasing expression levels of miR-378. As known, miR-378 is embedded within PPARGC1b, which encodes PGC-1β, and could be stimulated by feeding lauric acid in cells. However, lauric acid is a type of saturated fatty acid, and therefore daily intake in humans is no recommended. Consequently, we tried to replace it with eicosapentaenoic acid (EPA or also icosapentaenoic acid), an omega-3 unsaturated fatty acid that is FDA-approved. Following this, we modulated the expression level of miR-378 in KRAS or BRAF mutants by feeding EPA in vitro, and eventually revealed the mechanism of cancer cells remission that currently remains unknown. The western blotting and ELISA assay were performed to analyze the MAPK/ERK pathway and caspase pathway in three cell lines (SW480 and HCT116 with contain KRAS mutants, and HT29 with contain BRAF mutants), including cell lines before and after feeding with 40 μM concentration of EPA. All experiments were then compared to the wild type cells (caco2), the control cell line. The results showed higher expression of miR-378 in all types of mutated cells, except the wild type CRC cells. In addition, lower cancer cell survival rate was observed in cells that were given 0.2 μM of cetuximab treatment, especially in KRAS mutated cells as well as control wild type cells (p = 0.010∼0.013). The total ERK1/2 protein in the KRAS mutant CRC cells and wild type CRC cell showed lower expression levels after receiving 40 μM EPAee for 24 hours (p = 0.022∼0.035). A higher phosphorylated proteins status of ERK1/2 was also seen (p = 0.006∼0.047). However, the opposite result was noted in BRAF mutant cells. In cells further treated with anti-EGFR antibody for 48 hours, cell viability was significantly lowered in KRAS mutant and control wild type cells (p = 0.006∼0.013), but there was no sensitivity of anti-EGFR antibody reaction to the EPA-fed BRAF mutant cell. The data demonstrated with EPA may indeed significantly induce the expression of miR-378, and further restored the sensitivity of anti-EGFR antibody in KRAS mutant cells. In conclusion, KRAS mutant cells may restore sensitivity to cetuximab after up-regulation of the miR-378 induced by EPA. This finding has offered a new alternative therapeutic solution for future patients suffering from KRAS mutated colorectal cancer. Citation Format: Chih-Wei Chen, Li-Wei Kuo, Yeu-Jye Pang, Wai-Hung Leung, Wen-Hui Weng. Using eicosapentaenoic acid to improve cetuximab sensitivity in KRAS mutants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2695. doi:10.1158/1538-7445.AM2015-2695