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Supplementary Figure 1 from Amiloride Modulates Alternative Splicing in Leukemic Cells and Resensitizes Bcr-AblT315I Mutant Cells to Imatinib

The antihypertensive drug amiloride is being considered as a tactic to improve cancer therapy including that for chronic myelogenous leukemia. In this study, we show that amiloride modulates the alternative splicing of various cancer genes, including Bcl-x, HIPK3, and BCR/ABL, and that this effect is not mainly related to pH alteration, which is a known effect of the drug. Splice modulation involved various splicing factors, with the phosphorylation state of serine-arginine–rich (SR) proteins also altered during the splicing process. Pretreatment with okadaic acid to inhibit protein phosphatase PP1 reversed partially the phosphorylation levels of SR proteins and also the amiloride-modulated yields of Bcl-xs and HIPK3 U(-) isoforms. Genome-wide detection of alternative splicing further revealed that many other apoptotic genes were regulated by amiloride, including APAF-1, CRK, and SURVIVIN. Various proteins of the Bcl-2 family and MAPK kinases were found to be involved in amiloride-induced apoptosis. Moreover, the effect of amiloride on mRNA levels of Bcl-x was demonstrated to translate to the protein levels. Cotreatment of K562 and BaF3/Bcr-AblT315I cells with amiloride and imatinib induced more loss of cell viability than either agent alone. Our findings suggest that amiloride may offer a potential treatment option for chronic myelogenous leukemia either alone or in combination with imatinib. Cancer Res; 71(2); 383–92. ©2011 AACR.

Renal cell carcinoma (RCC) has emerged as a metabolic disease characterized by dysregulated expression of metabolic enzymes. Patients with metastatic RCC have an unusually poor prognosis and near-universal resistance to all current therapies. To ultimately improve cancer treatment and survival rate, there is an urgent need to reveal the mechanisms by which metabolic enzymes and aberrant pathways regulate oncogenic signaling. Through an integrated two-step analysis of RCC metabolic pathways, we previously identified dysregulated expression of methylthioadenosine phosphorylase (MTAP) in aggressive RCC. An accumulation of cellular methylthioadenosine has been observed in MTAP-deleted cancer cells. Interestingly, we found a decreased of protein-methylation level with concomitant with an increase in tyrosine phosphorylation after MTAP knockout. Next, we performed a phospho-kinase antibody array screen and identified the type 1 insulin-like growth factor-1 receptor (IGF1R) as the top-one candidate with upregulated tyrosine phosphorylation in response to MTAP loss. IGF1R phosphorylation acted upstream of Src and STAT3 signaling in MTAP-knockout RCC cells. Reduction of phospho-IGF1R by a selective inhibitor of IGF-1R, linsitinib, inhibited the cell migration and invasion capability of MTAP-deleted cells. Data from cell viability assays showed that MTAP loss enhanced linsitinib-mediated cytotoxicity in RCC cells. Our data suggest that IGF1R signaling is a driver pathway to confer aggrieves nature of MTAP-deleted renal cell carcinomas. Citation Format: Jihao Xu, Wen-Hsin Chang, Carissa Huang, David Yang, Ching-Hsien Chen. Targeting the insulin-like growth factor-1 receptor in MTAP-deleted renal cell carcinomas [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 3507.

Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/VEGF signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably VEGF-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multi-kinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multi-kinase inhibitors. Citation Format: Ching-Hsien Chen, Wen-Hsin Chang, Eric Yu, Muhammad S. Arif, Reen Wu, Robert H. Weiss. Dysregulation of myristoylated alanine-rich C kinase substrate (MARCKS): A novel therapeutic target in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2017-1810

Genome instability, metabolic switch, and activating metastasis are three of the cancer hallmarks, which always collectively contribute to cancer malignancy. We identified methylthioadenosine phosphorylase (MTAP) as a metastasis suppressor in a series isogenic lung cell lines with different invasion ability by array CGH. Using siRNA silencing and ectopic expression of MTAP, we found that MTAP not only inhibited invasion and colony forming in vitro, but also reduced metastasis and tumorigenesis in vivo. Clinically, patients with low MTAP expression associated with poor overall survival and progression-free survival. Furthermore, the metabolite analysis of clinical specimens showed that low MTAP expression leads to MTA substrate accumulation and product decrease. MTA accumulation in MTAP knockout cells reduced the level of symmetric arginine dimethylation of proteins. By using LC/MS, we identified a novel methyl-protein, whose level is elevated in MTAP knockout cells with the depletion of symmetric arginine dimethylation. Functionally, the unmethylated protein would be protected from proteolysis via caspase 3 cleavage. Moreover, the unmethylated mutant protein promoted migration and invasion in vitro strongly than the wild-type one did. In conclusion, we identify a novel marker in low MTAP expression or MTAP knockout cells, providing a potential therapeutic candidate in lung adenocarcinoma. Citation Format: Wen-Hsin Chang, Bing-Ching Ho, Kang-Yi Su, Jian-Wei Chen, Reen Wu, Pan-Chyr Yang, Sung-Liang Yu. MTAP insufficiency promotes metastasis in lung cancer via diminishing protein dimethylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1544. doi:10.1158/1538-7445.AM2017-1544

Increasing evidence had suggested important roles of protein kinase C (PKC)-mediated phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in modulating various cellular processes, including cancer cell growth and metastasis. However, there is a lack of information regarding to the roles of MARCKS and its pSer 159/163 phosphorylated product in breast cancer cells and tissues. To tackle this concern, we recently initiated both immunohistochemical and western blot analyses on a series of clinical specimens and several breast cancer cell lines. We have observed a significant elevation of phospho-MARCKS associated with advanced-stage breast cancer tissues as compared with benign ones, particularly in triple-negative breast cancer (TNBC) tissues and cell lines. Knockdown of MARCKS expression resulted in suppressions of lamellipodia/filopodia formation and migration/invasion as well as down-regulation of Src activity in these TNBC cell lines. Of note, MARCKS-silenced TNBC cells grew much more slowly and more sensitive to paclitaxel than the control and parental cells. Treatment with paclitaxel was shown to induce MARCKS phosphorylation in a dose-dependent manner, which may be related to an enhanced paclitaxel resistance in some of TNBC cells. However, PKC inhibitors were able to abrogate paclitaxel-induced MARCKS phosphorylation. Consistent with this potential, treatments of TNBC cells with a MARCKS N-terminus sequence peptide, namely MANS, to down regulate PKC/MARCKS pathway not only attenuated the associated aggressive phenotype but also synergistically enhanced paclitaxel-induced cytotoxicity in vitro and in vivo. These results demonstrate an association of PKC-mediated MARCKS phosphorylation with breast cancer malignancy potential and MARCKS phosphorylation as a predictor of paclitaxel resistance in TNBC cells. It is suggestive that an inhibition of PKC/MARCKS pathway may serve as an alternative therapeutic strategy for enhancing efficacy of chemotherapy. Citation Format: Ching-Hsien Chen, Muhammad Arif, Wen-Hsin Chang, Yuan Yuan, Jing Zhai, David K. Ann, Reen Wu. PKC/MARCKS pathway is a novel therapeutic target associated with breast cancer malignancy potential and paclitaxel resistance. [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 2769. doi:10.1158/1538-7445.AM2014-2769

14-3-3 family consists of seven highly conserved isoforms and most of them are identified as oncogenes in various types of cancer except for 14-3-3σ, a well-known tumor suppressor. To clarify the suppressor characteristic of 14-3-3σ divergent from others, we found that the major difference between the other oncogenic members and 14-3-3σ by protein alignment was an amino acid substitution (Y180H) by which the SH2-binding motif (YYEI) is disrupted and cannot be phosphorylation. Thus, we generated a H180Y 14-3-3σ mutant and investigated the impact of the acquired YYEI motif on the tumor suppression. First, we found that 14-3-3σ decreased cancer invasion. Surprisingly, the H180Y mutant not only enhanced cell invasion but increased cell viability. Meanwhile, the interaction between H180Y mutant and Src was higher than wild type. It indicates one amino acid substitution switches 14-3-3σ from tumor suppressor to oncogene. Hence, the YYEI motif might be important for the oncogenicity of other 14-3-3 proteins. Indeed, we demonstrated that 14-3-3ζ interacted with Src through Y178 phosphorylation, which is crucial for the binding of 14-3-3ζ with Src-SH2 domain. Owing to the importance of Y178 phosphorylation in the 14-3-3ζ/Src interaction, we introduced the Y178F 14-3-3ζ mutant to lung cancer cells and confirmed that Y178 phosphorylation is important for the increase of invasion and viability. Furthermore, we also revealed an amino acid switch of 14-3-3σ from YYEI to HYEI during evolutionary progression. Taken together, our findings suggest that YYEI motif is essential for 14-3-3 proteins to interact with Src and to regulate Src-mediated cell functions. Citation Format: Wen-Hsin Chang, Ching-Hsien Chen, Qi-Sheng Hong, Jian-Wei Chen, Sung-Liang Yu, Chiung-Tong Chen. YYEI motif is critical to oncogenicity of 14-3-3 proteins. [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 3527. doi:10.1158/1538-7445.AM2014-3527