Shih-Shin Chang

Signaling cross-talk in the resistance to HER family receptor targeted therapy

Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) have an important role in the initiation and progression of various types of cancer. Inhibitors targeting these receptor tyrosine kinases are some of the most successful targeted anticancer drugs widely used for cancer treatment; however, cancer cells have mechanisms of intrinsic and acquired drug resistance that pose as major obstacles in drug efficacy. Extensive studies from both clinical and laboratory research have identified several molecular mechanisms underlying resistance. Among them is the role of signaling cross-talk between the EGFR/HER2 and other signaling pathways. In this review, we focus particularly on this signaling cross-talk at the receptor, mediator and effector levels, and further discuss alternative approaches to overcome resistance. In addition to well-recognized signaling cross-talk involved in the resistance, we also introduce the cross-talk between EGFR/HER2-mediated pathways and pathways triggered by other types of receptors, including those of the Notch, Wnt and TNFR/IKK/NF-κB pathways, and discuss the potential role of targeting this cross-talk to sensitize cells to EGFR/HER2 inhibitors.

Aurora A kinase activates YAP signaling in triple-negative breast cancer

The Yes-associated protein (YAP) is an effector that transduces the output of the Hippo pathway to transcriptional modulation. Considering the role of YAP in cancers, this protein has emerged as a key node in malignancy development. In this study, we determined that Aurora A kinase acts as a positive regulator for YAP-mediated transcriptional machinery. Specifically, YAP associates with Aurora A predominantly in the nucleus. Activation of Aurora A can impinge on YAP activity through direct phosphorylation. Moreover, aberrant expression of YAP and Aurora A signaling is highly correlated with triple-negative breast cancer (TNBC). We herein provide evidence to establish the functional relevance of this newly discovered regulatory axis in TNBC.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformins role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Abstract B03: Cisplatin-resistant cells possess collateral sensitivity to folate antimetabolites

Cisplatin is widely used as a first-line chemotherapeutic agent for lung cancers. Although effective initially, lung cancer cells eventually acquire resistance to cisplatin, causing tumor recurrence. Understanding mechanisms that govern cisplatin resistance is expected to improve therapeutic efficacy in lung cancer patients. Although various studies using cisplatin-resistant cell lines have revealed possible mechanisms of resistance, the applicability of these mechanisms in a clinical setting are debatable. One possible reason for this discrepancy may be the use of cell line models which are exposed to unnaturally high doses of cisplatin to yield cells with a resistant phenotype. These cell lines may not be representative of naturally arising cisplatin-resistant cells in tumors that are treated according to a clinically established dose and schedule. Therefore, to improve the experimental model, we have established two new cisplatin-resistant lung cancer cell lines through intermittent low dose challenges that mimic a clinical treatment schedule (one pulse/month for up to six cycles). The resistant cell lines resulting from this procedure demonstrate improved clonogenic potential and sphere-forming properties, suggesting an increase in the population of tumor-initiating cells. Interestingly, via testing with several different anti-cancer drugs, we found that these resistant cells are more sensitive to the antifolate Pemetrexed, in comparison to their parental cells. To conclude, our study suggests that sequential administration of antifolates might be useful in the treatment of lung cancers that have acquired cisplatin resistance. Citation Format: Shih-Shin Chang, Hirohito Yamaguchi, Mien-Chie Hung. Cisplatin-resistant cells possess collateral sensitivity to folate antimetabolites. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B03.

Abstract A49: S6K1 promotes invasiveness of breast cancer cells in a novel model of triple-negative breast cancer metastasis

Introduction: Breast cancer is the second-leading cause of oncology-related death in US women. Despite extensive research, over 30% of breast cancer patients develop metastatic disease, and metastases account for majority of breast cancer-associated morbidity and mortality. Of all invasive breast cancers, patients with tumors lacking expression of the Estrogen and Progesterone hormone Receptors and Human Epidermal growth factor Receptor 2 have the poorest clinical prognosis. These triple-negative tumors (TNBC) represent an aggressive form of the disease that is marked by an early-onset metastasis, a high tumor recurrence rate, and a low overall survival during the first three years post-diagnosis. However, few TNBC mouse models of metastasis currently exist. Results: We noticed that a well-established MDA-MB-231 TNBC cell line produces rapid and extremely lytic bone lesions in lumbar, sacral and caudal vertebrae, and hind limbs in about 10% of animals injected intravenously. We biopsied one of these bone metastases and established a new metastatic MDA-MB-231-1566 cell line. Following an intravenous injection, MDA-MB-231-1566 cells produce early-onset metastasis to bone in up to 70% of animals with concurrent metastases to lungs, liver, and soft tissues. We demonstrate that 100% of animals injected with MDA-MB-231-1566 cells developed metastasis and had median survival of 60 days vs. 80 days in mice injected with the parental cell line. We also demonstrate that ribosomal protein S6 is highly phosphorylated on Ser235/236 in metastatic TNBC tumors, and that this phosphorylation is indicative of upstream S6 kinase (S6K) activity. Lastly, we provide evidence that targeting S6K with a highly specific inhibitor, PF-4708671, at sub-lethal doses inhibits cell migration without inducing cell death, and thus may provide a potent anti-metastatic adjuvant therapy approach. Conclusion: We established a new model of rapid TNBC metastases to multiple organs following a simple intravenous injection. We believe this model provides a valuable tool for screening new therapeutics aimed to stop growth of metastases. Citation Format: Yekaterina B. Khotskaya, Aarthi Goverdhan, Jia Shen, Mariano Ponz Sarvice, Shih-Shin Chang, Ming-Chuan Hsu, Yongkun Wei, Weiya Xia, Patricia Steeg, Dihua Yu, Mien-Chie Hung. S6K1 promotes invasiveness of breast cancer cells in a novel model of triple-negative breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A49.

Abstract 3027: A novel function of EGFR in epigenetic modulation via hMOF in breast cancer .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnThe epidermal growth factor receptor (EGFR) is aberrantly active in breast cancer, initiates and drives tumorigenic pathways, and has been implicated in multiple tumorigenic processes including hyperplasia, chemo-resistance and metastasis. Histone lysine modifications directly influence activation and repression of transcription. MYST family acetyltransferase, human males absent on the first (hMOF), plays an important role in transcription activity through histone H4K16 acetylation. Loss of hMOF and subsequently the absence of H4K16 acetylation have been shown to be a common feature in human cancer including breast cancer. However, the regulation of hMOF remains largely unknown. Recently, we have discovered the acetylation of EGFR in human breast cancer cells. Thus, we screened a set of EGFR-interacting acetyltransferases using co-immunoprecipitation analysis and ingenuity pathway analysis (IPA). Here, we found that EGFR directly interacts with and phosphorylates hMOF. EGFR-induced phosphorylation hMOF was significantly reduced in the hMOF YF mutant. Indeed, hMOF YF mutant showed a higher histone acetyltransferase (HAT) activity compare to wild type hMOF. In addition, tyrosine kinase inhibitor (TKI) also enhanced the HAT activity of wild type hMOF. Furthermore, we found that EGFR modulates acetylation status of histone H4K16 via phosphorylating hMOF. We anticipate our study of EGFR and hMOF to be of therapeutic relevance, possibly leading to novel discovery in drug development targeting the receptor tyrosine kinase and the acetylation process.nnCitation Format: Seung-Oe Lim, Chia-Wei Li, Shih-Shin Chang, Jia Shen, Mien-Chie Hung. A novel function of EGFR in epigenetic modulation via hMOF in breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2013-3027

Abstract 1051: EGFR inhibitors attenuate caspase-independent cell death and confer negative effects on cisplatin .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnEGFR tyrosine kinase inhibitors (TKIs) have demonstrated exciting results and led to advanced therapy trials in combination with chemotherapy. However, clinical studies have raised an important question why the antagonism between TKI and chemotherapy occurs in lung cancer patients only with wildtype but not mutant EGFR. In this study, we further explored the mechanism through which gefitinib gives rise to the negative effects when combined with cisplatin in wildtype EGFR lung cancer cells. We found that gefitinib inhibited cisplatin-induced caspase-independent cell death (CID) but not caspase-dependent cell death (apoptosis). In wildtype EGFR cells, gefitinib not only inhibited CID but also failed to induce apoptosis and therefore compromising the efficacy of cisplatin. Moreover, inhibition of the EGFR/ERK/AKT pathway by gefinitib activated FOXO3a and subsequently reduced reactive oxygen species (ROS) induced by cisplatin treatment and ROS-mediated CID. To overcome this, we showed that SAHA and erastin, which are known to induce ROS-mediated CID, strongly enhanced the effect of cisplatin in wildtype EGFR cells. Taken together, our findings provide a possible explanation for the previously failed clinical trials as well as a novel insight into future cancer therapy.nnCitation Format: Hirohito Yamaguchi, Jennifer L. Hsu, Chun-Te Chen, Ying-Nai Wang, Ming-Chuan Hsu, Shih-Shin Chang, Yi Du, How-Wen Ko, Roy S. Herbst, Mien-Chie Hung. EGFR inhibitors attenuate caspase-independent cell death and confer negative effects on cisplatin . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1051. doi:10.1158/1538-7445.AM2013-1051

Abstract 5351: Phosphorylation of EZH2 at Thr487 is a marker for good prognosis in pancreatic cancer patients.

EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 is overexpressed in multiple tumor types and correlates with poor prognosis. We have previous shown that phosphorylation of EZH2 at Thr 487 by CDK1 inhibits its methyltransferase activity. However, the status of phosphorylation of EZH2 at Thr 487 (P-EZH2-T487) and its clinical implication in cancer patients have not been reported. We thus examined P-EZH2-T487 by immunohistochemistry and its association with prognosis in pancreatic cancer patients. We found that P-EZH2-T487 can be seen in 61% (47/77) of pancreatic cancer. In univariate survival analysis, patients with high P-EZH2-T487 had significantly longer overall survival time when compared with those with low P-EZH2-T487 (P=0.032). In multivariate analysis, P-EZH2-T487 was an independent prognostic marker for overall survival in pancreatic cancer (HR=0.358, P=0.001). These results suggest that P-EZH2-T487 is a prognostic indicator for clinical outcome in pancreatic cancer patients. Citation Format: Yongkun Wei, Shih-Shin Chang, Huamin Wang, Weiya Xia, Mien-Chie Hung. Phosphorylation of EZH2 at Thr487 is a marker for good prognosis in pancreatic cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5351. doi:10.1158/1538-7445.AM2013-5351 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3290: A novel model of breast cancer metastasis: Killing two birds with one stone

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnAmerican women carry a lifetime risk of 1:8 of developing breast cancer, with metastatic disease remaining incurable. Between 70-80% of breast cancer patients diagnosed with metastases will exhibit lesions in their skeleton and in 25% of patients bone is the first site of metastasis. Breast cancer bone metastases are most frequently osteolytic. Thus, patients exhibiting bone lesions might suffer from nerve compression, bone fractures, hypercalcemia, pain, or paralysis. Existing treatment strategies for patients with bone metastases are palliative and designed to limit cancer-associated bone loss. It has been difficult to study breast cancer dissemination to bone due to lack of good animal models and little biopsy tissue availability. For human xenografts to establish bone lesions, human breast cancer cells are injected into the left ventricle of the heart, a procedure technically challenging and not 100% efficient. Resulting bone lesions are slow to develop and may be easy to miss on x-ray examination. Here, we show that a well-described parental MDA-MB-231 breast cancer cell line recapitulates progression of human disease when injected intravenously through the lateral tail vein into athymic nude mice. For reasons yet unclear, 30-50% of animals develop rapid and extremely lytic bone lesions in lumbar, sacral and caudal vertebrae and hind limbs. Similar to breast cancer patients, these animals exhibit pathologic fractures and paralysis, necessitating euthanasia within 4 weeks after cell inoculation. Interestingly, animals not affected by bone metastases go on to develop overt lung metastases within 10-12 weeks after cell inoculation. Bioluminescence imaging studies indicated presence of luciferase-tagged MDA-MB-231 cells in lungs of all mice at 2 and 24 hours post-cell inoculation. Within three weeks post-injection, large lesions were detected in the lumbar to caudal vertebrae regions and hind limbs of mice injected with luciferase-tagged (6/15 mice) or parental MDA-MB-231 cells (7/13 mice). Bone osteolysis was confirmed by x-ray, and paralysis occurred as early as 3 weeks after cell injection. Cytogenetic “finger printing” preformed by the institutional core facility confirmed that cell lines used exhibit properties of MDA-MB-231 cells consistent with ATCC profile. Furthermore, molecular comparison of these cell lines with MDA-MB-231 cells obtained from other sources, including ATCC, revealed no differences. Unexpectedly, when examined in an orthotopic breast cancer model, MDA-MB-231 cells used in these studies produced rapidly growing tumors that spontaneously metastasized to lymph nodes and lungs, while MDA-MB-231 cells purchased from ATCC were completely non-tumorigenic. Studies are underway to determine a molecular mechanism responsible for this aggressive metastatic phenotype. This model may serve as a valuable tool for screening new therapeutics aimed to stop growth of metastases at multiple sites.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3290. doi:1538-7445.AM2012-3290