Wan-Lun Wang

Sorafenib analogue SC‐60 induces apoptosis through the SHP‐1/STAT3 pathway and enhances docetaxel cytotoxicity in triple‐negative breast cancer cells

Recurrent triple‐negative breast cancer (TNBC) needs new therapeutic targets. Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) can act as a tumor suppressor by dephosphorylating oncogenic kinases. One major target of SHP‐1 is STAT3, which is highly activated in TNBC. In this study, we tested a sorafenib analogue SC‐60, which lacks angiokinase inhibition activity, but acts as a SHP‐1 agonist, in TNBC cells. SC‐60 inhibited proliferation and induced apoptosis by dephosphorylating STAT3 in both a dose‐ and time‐dependent manner in TNBC cells (MDA‐MB‐231, MDA‐MB‐468, and HCC1937). By contrast, ectopic expression of STAT3 rescued the anticancer effect induced by SC‐60. SC‐60 also increased the SHP‐1 activity, but this effect was inhibited when the N‐SH2 domain (DN1) was deleted or with SHP‐1 point mutation (D61A), implying that SHP‐1 is the major target of SC‐60 in TNBC. The use of SC‐60 in combination with docetaxel synergized the anticancer effect induced by SC‐60 through the SHP‐1/STAT3 pathway in TNBC cells. Importantly, SC‐60 also displayed a significant antitumor effect in an MDA‐MB‐468 xenograft model by modulating the SHP‐1/STAT3 axis, indicating the anticancer potential of SC‐60 in TNBC treatment. Targeting SHP‐1/p‐STAT3 and the potential combination of SHP‐1 agonist with chemotherapeutic docetaxel is a feasible therapeutic strategy for TNBC.

Sequential combination of docetaxel with a SHP-1 agonist enhanced suppression of p-STAT3 signaling and apoptosis in triple negative breast cancer cells

Triple negative breast cancer (TNBC) is an aggressive cancer for which prognosis remains poor. Combination therapy is a promising strategy for enhancing treatment efficacy. Blockade of STAT3 signaling may enhance the response of cancer cells to conventional chemotherapeutic agents. Here we used a SHP-1 agonist SC-43 to dephosphorylate STAT3 thereby suppressing oncogenic STAT3 signaling and tested it in combination with docetaxel in TNBC cells. We first analyzed messenger RNA (mRNA) expression of SHP-1 gene (PTPN6) in a public TNBC dataset (TCGA) and found that higher SHP-1 mRNA expression is associated with better overall survival in TNBC patients. Sequential combination of docetaxel and SC-43 in vitro showed enhanced anti-proliferation and apoptosis associated with decreased p-STAT3 and decreased STAT3-downstream effector cyclin D1 in the TNBC cell lines MDA-MB-231, MDA-MB-468, and HCC-1937. Ectopic expression of STAT3 reduced the increased cytotoxicity induced by the combination therapy. In addition, this sequential combination showed enhanced SHP-1 activity compared to SC-43 alone. Furthermore, the combination treatment-induced apoptosis was attenuated by small interfering RNA (siRNA) against SHP-1 or by ectopic expression of SHP-1 mutants that caused SC-43 to lose its SHP-1 agonist capability. Moreover, combination of docetaxel and SC-43 showed enhanced tumor growth inhibition compared to single-agent therapy in mice bearing MDA-MB-231 tumor xenografts. Our results suggest that the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxicity by SHP-1 dependent STAT3 inhibition in human triple negative breast cancer cells. TNBC patients with high SHP-1 expressions show better survival. Docetaxel combined with SC-43 enhances cell apoptosis and reduces p-STAT3. SHP-1 inhibition reduces the enhanced effect of docetaxel-SC-43 combination. Docetaxel-SC-43 combination suppresses xenograft tumor growth and reduces p-STAT3.Key messagesTNBC patients with high SHP-1 expressions show better survival.Docetaxel combined with SC-43 enhances cell apoptosis and reduces p-STAT3.SHP-1 inhibition reduces the enhanced effect of docetaxel-SC-43 combination.Docetaxel-SC-43 combination suppresses xenograft tumor growth and reduces p-STAT3.

Overexpression of phosphoprotein phosphatase 2A predicts worse prognosis in patients with breast cancer: a 15-year follow-up

Breast cancer subtypes can be stratified by IHC expression of estrogen receptor, progesterone receptor, and human epidermal growth factor 2 (HER2). The signaling pathways mediated by these receptors are the dominant drivers of cell proliferation and survival in most human breast cancers. One of the most frequently overactivated pathways in breast cancer is the AKT signaling cascade. Protein phosphatase 2A (PP2A) acts as a switch to turn off signal transduction in the AKT pathway; however, it is frequently inactivated in many cancers by phosphorylation of Tyr-307 to form phosphoprotein phosphatase 2A (p-PP2A). This study aimed to investigate the clinical significance of p-PP2A and phospho-AKT (p-AKT) expression in 672 patients with breast cancer during a 15-year follow-up. The breast tissue microarray was evaluated for p-PP2A and p-AKT expression using IHC staining and scores. Analysis of IHC staining results revealed that p-PP2A expression was positively correlated with HER2, Ki-67, and p-AKT overexpression (P<.001, P=.003, and P=.001, respectively). At the time of diagnosis, breast cancer patients with higher p-PP2A expression had significantly shorter 15-year OS than patients with lower p-PP2A expression did (P=.017). Multivariate Cox regression analysis revealed that high p-PP2A expression was an independent prognostic factor for shorter OS (hazard ratio, 1.741; P=.012). Our data revealed that high p-PP2A expression is positively associated with HER2, Ki-67, and p-AKT expression. High p-PP2A expression correlates with poor clinical outcomes in breast cancer, especially in patients with TNBC.

SET Overexpression is Associated with Worse Recurrence-Free Survival in Patients with Primary Breast Cancer Receiving Adjuvant Tamoxifen Treatment

Adjuvant tamoxifen reduces the recurrence rate of estrogen receptor (ER)-positive breast cancer. Previous in vitro studies have suggested that tamoxifen can affect the cancerous inhibitor of protein phosphatase 2A (CIP2A)/protein phosphatase 2A (PP2A)/phosphorylation Akt (pAkt) signaling in ER-negative breast cancer cells. In addition to CIP2A, SET nuclear proto-oncogene (SET) oncoprotein is another intrinsic inhibitor of PP2A, participating in cancer progression. In the current study, we explored the clinical significance of SET, CIP2A, PP2A, and Akt in patients with ER-positive breast cancer receiving adjuvant tamoxifen. A total of 218 primary breast cancer patients receiving adjuvant tamoxifen with a median follow-up of 106 months were analyzed, of which 17 (7.8%) experienced recurrence or metastasis. In an immunohistochemical (IHC) stain, SET overexpression was independently associated with worse recurrence-free survival (RFS) (hazard ratio = 3.72, 95% confidence interval 1.26–10.94, p = 0.017). In silico analysis revealed mRNA expressions of SET, PPP2CA, and AKT1 significantly correlated with worse RFS. In vitro, SET overexpression reduced tamoxifen-induced antitumor effects and drove luciferase activity in an Estrogen receptor element (ERE)-dependent manner. In conclusion, SET is a prognostic biomarker in patients with primary ER-positive breast cancer receiving adjuvant tamoxifen and may contribute to the failure of the tamoxifen treatment by modulating the ER signaling. Our study warrants further investigation into the potential role of SET in ER-positive breast cancer.

Carfilzomib induces leukaemia cell apoptosis via inhibiting ELK1/KIAA1524 (Elk-1/CIP2A) and activating PP2A not related to proteasome inhibition.

Enhancing the tumour suppressive activity of protein phosphatase 2A (PP2A) has been suggested to be an anti‐leukaemic strategy. KIAA1524 (also termed CIP2A), an oncoprotein inhibiting PP2A, is associated with disease progression in chronic myeloid leukaemia and may be prognostic in cytogenetically normal acute myeloid leukaemia. Here we demonstrated that the selective proteasome inhibitor, carfilzomib, induced apoptosis in sensitive primary leukaemia cells and in sensitive leukaemia cell lines, associated with KIAA1524 protein downregulation, increased PP2A activity and decreased p‐Akt, but not with the proteasome inhibition effect of carfilzomib. Ectopic expression of KIAA1524, or pretreatment with the PP2A inhibitor, okadaic acid, suppressed carfilzomib‐induced apoptosis and KIAA1524 downregulation in sensitive cells, whereas co‐treatment with the PP2A agonist, forskolin, enhanced carfilzomib‐induced apoptosis in resistant cells. Mechanistically, carfilzomib affected KIAA1524 transcription through disturbing ELK1 (Elk‐1) binding to the KIAA1524 promoter. Moreover, the drug sensitivity and mechanism of carfilzomib in xenograft mouse models correlated well with the effects of carfilzomib on KIAA1524 and p‐Akt expression, as well as PP2A activity. Our data disclosed a novel drug mechanism of carfilzomib in leukaemia cells and suggests the potential therapeutic implication of KIAA1524 in leukaemia treatment.

Abstract 3065: Erlotinib derivative, devoid of EGFR kinase inhibiting effect, induced apoptosis of triple-negative breast cancer cells through modulating Elk-1/CIP2A signaling pathway

Background: Triple-negative breast cancer (TNBC), a subgroup of breast cancer, is characterized by its aggressive nature and poor prognosis. Therefore, the discovery and identification of new therapeutic molecule is currently one of urgent issues. Previous studies have suggested an oncoprotein, cancerous inhibitor of protein phosphatase 2A (CIP2A), is a potential therapeutic determinant that mediates the anti-cancer effects of several new agents. We have developed an erlotinib analogue TD-19 that is devoid of EGFR tyrosine kinase inhibition effect, and demonstrated its anti-tumor effects through the suppression of CIP2A-mediated pathway in non-small-cell lung cancer cells (J Pharmacol Exp Ther 2014). In this study, we tested the apoptotic effect of TD-19 in TNBC cells and examined the drug mechanism upstream of CIP2A. Methods: TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of TD-19 was tested in nude mice with breast cancer xenografts. Results: TD-19 triggered significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-468 and BT-20 cell lines, as well as in non-TNBC cell lines (SK-BR3 and MCF-7). The induced apoptotic effects are associated with CIP2A and p-Akt downregulation in both dose- and time-dependent manners. Overexpression of CIP2A protected MDA-MB-468 from the induced apoptosis of TD-19. In addition, PP2A activity was increased in TD-19 treated MDA-MB-468 cells. Moreover, forskolin, a protein phosphatase 2A (PP2A) activator, enhanced the cell death induced by TD-19, whereas okadaic acid, a PP2A antagonist, attenuated the apoptosis. Mechanistically, we demonstrated that TD-19 downregulated CIP2A mRNA expression through affecting the total amount of Elk-1 in nucleus. Chromatin immunoprecipitation experiments showed TD-19 disturbed the binding of Elk-1 to the proximal CIP2A-promotor. Furthermore, TD-19 showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and downregulated CIP2A as well as p-Akt in these xenografted tumors. Interestingly, combining TD-19 with cisplatin demonstrated enhanced anti-proliferative and apoptotic effects in association with CIP2A downregulation in vitro. Conclusions: Our results suggest that the novel erlotinib derivative (TD-19) induced apoptosis through inhibiting Elk1/CIP2A/PP2A/p-Akt signaling in TNBC cells. Pharmacological modulation of Elk-1/CIP2A signaling may be a therapeutic strategy for TNBC. (Supported by Yen Tjing Ling Medical Foundation (CI-104-07); MOST 104-2628-B-075-001-MY3; Yang-Ming Branch of Taipei City Hospital; and from the Ministry of Health and Welfare, Executive Yuan, Taiwan, MOHW104-TDU-B-211-124-001; and MOHW103TDU-212-114002). Citation Format: Ling-Ming Tseng, Yi-Ting Chen, Chun-Teng Huang, Pei-Yi Chu, Wan-Lun Wang, Chun-Yu Liu, Chung-Wai Shiau, Kuen-Feng Chen. Erlotinib derivative, devoid of EGFR kinase inhibiting effect, induced apoptosis of triple-negative breast cancer cells through modulating Elk-1/CIP2A signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3065.

Abstract 1261: Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway

Background: Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive subtype that currently lack of specific targeted therapy. Previous studies have suggested that disturbing the oncogenic STAT3 signaling by activating a protein tyrosine phosphatase SHP-1 as a possible anti-cancer strategy. Our previous study also found that nintedanib (BIBF1120), a multi-kinase inhibitor that blocks mainly anti-angiogenic pathways and has been approved for idiopathic pulmonary fibrosis and for non-small cell lung cancer (in Europe), inhibits hepatocellular carcinoma cell growth independent of its anti-angiokinase activity (J Hepatology 2015). In this study, we tested nintedanib in TNBC cells and examined the underlying molecular mechanism by which nintedanib shows anti-TNBC effects. Methods: TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC-1395) were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. Purified SHP-1 proteins or cells expressing deletion N-SH2 domain or D61A point mutants (SHP-1 mutants with constant open form conformations) were used to investigate the potential effects of nintedanib on SHP-1. In vivo efficacy of nintedanib was tested in nude mice bearing orthotopic breast cancer cells xenografts. Results: MTT assays revealed nintedanib induced anti-proliferation in the 3 TNBC cell lines. Sub-G1 flow cytometric and western blot analysis showed that nintedanib induced apoptosis in association with downregulation of p-STAT3 and its downstream proteins such as cyclin D1 in both dose and time-dependent manners. Overexpression of STAT3 suppressed nintedanib-mediated apoptosis in MDA-MB-231 cells. Nintedanib further increased SHP-1 activity in purified SHP-1 proteins, suggesting that nintedanib may directly affects SHP-1 activity for p-STAT3 inhibition. Moreover, either specific SHP-1 activity inhibitor or SHP-1 siRNA reduced the apoptotic effects of nintedanib on MDA-MB-231 and MDA-MB-468 cells, validating the role of SHP-1 activation in nintedanib-induced apoptosis. Furthermore, nintedanib-induced apoptosis was attenuated in TNBC cells expressing SHP-1 mutants with constant open form conformations, including deletion mutants of N-SH2 domain or D61A mutants, suggesting that the auto-inhibition structure of SHP-1 mediated the effect of nintedanib. Importantly, intraperitoneal injection of nintedanib showed antitumor activity, increased SHP-1 activity and p-STAT3 downregulation in MDA-MB-231 xenograft tumors. Conclusions Our results suggest that nintedanib induced anti-tumor effect by SHP-1 dependent STAT3 inhibition in human TNBC cells. These findings may provide new signaling pathway insight into the inhibition of TNBC growth by nintedanib. Citation Format: Chun-Yu Liu, Chia-Han Lee, Chun-Teng Huang, Pei-Yi Chu, Wan-Lun Wang, Ling-Ming Tseng, Chung-Wai Shiau, Kuen-Feng Chen. Nintedanib induces antitumor effects in triple-negative breast cancer cells by targeting SHP-1/p-STAT3-mediated signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1261.

Abstract 4465: Sequential combination of docetaxel with a novel SHP-1 agonist enhanced anti-tumor effect in triple negative breast cancer cells

Background: Interfering the oncogenic STAT3 signaling is considered as promising anti-cancer strategy. STAT3 signaling can be negatively regulated by the protein tyrosine phosphatase SHP-1 through direct dephosphorylation of p-STAT3 (Tyr705). Our previous studies have confirmed SC-43 as a novel SHP-1 enhancer which down-regulate p-STAT3 and STAT3-related downstream effectors (Breast Cancer Research 2013). Here, we tested the efficacy of SC-43 in combination with docetaxel in triple negative breast cancer cells. Methods: TNBC cell lines (HCC-1937, MDA-MB-468, MDA-MB-231) were used for in vitro studies. Cell viability was examined by MTT assay. Combination index was determined using Calcusyn anaysis. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of SC-43 in combination with docetaxel was tested in nude mice with breast cancer xenografts. Results: Sequential combination of docetaxel with SC-43 showed enhanced anti-proliferative activity in all tested TNBC cells. Synergistic effect was demonstrated when docetaxel combined with SC-43 at a dose ratio of 1 to 10, showing combination index below 1.0. Sub-G1 and western blot analysis showed that SC-43 enhanced docetaxel-induced apoptosis. In addition, combining SC-43 with docetaxel efficiently suppressed p-STAT3 and downstream effectors. Ectopic expression of STAT3 reduced the enhanced cytotoxicity by the combination therapy. Previous studies has shown SC-43 enhanced SHP-1 activity, here we showed that SC-43 in combination with docetaxel significantly enhanced SHP-1 activity. More importantly, intraperitoneal injection of SC-43 with tri-weekly docetaxel showed enhanced anti-tumor growth in xenografted tumor mice. Conclusion: Our results suggest that the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxic killing by SHP-1 dependent STAT3 inhibition in human triple negative breast cancer cells. (Supported by Yen Tjing-Ling Medical Foundation CI-104-07; NSC-102-2325-B-075-006 and MOST 103-2325-B-075-002; and MOHW103-TD-B-111-02) Citation Format: Ling-Min Tseng, Chun-Yu Liu, Shiu-Ping Yang, Wan-Lun Wang, Jung-Chen Su, Chia-Yun Wu, Chung-Wai Shiau, Kuen-Feng Chen. Sequential combination of docetaxel with a novel SHP-1 agonist enhanced anti-tumor effect in triple negative breast cancer cells. [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 4465. doi:10.1158/1538-7445.AM2015-4465