Tzu-Ting Huang

Alteration of SHP-1/p-STAT3 Signaling: A Potential Target for Anticancer Therapy

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1), a non-receptor protein tyrosine phosphatase, has been reported as a negative regulator of phosphorylated signal transducer and activator of transcription 3 (STAT3) and linked to tumor development. In this present review, we will discuss the importance and function of SHP-1/p-STAT3 signaling in nonmalignant conditions as well as malignancies, its cross-talk with other pathways, the current clinical development and the potential role of inhibitors of this pathway in anticancer therapy and clinical relevance of SHP-1/p-STAT3 in cancers. Lastly, we will summarize and highlight work involving novel drugs/compounds targeting SHP-1/p-STAT3 signaling and combined strategies that were/are discovered in our and our colleagues’ laboratories.

Treatment for the endocrine resistant breast cancer: Current options and future perspectives.

Endocrine resistance remains a challenge and an unmet need for managing hormone receptor-positive breast cancer. The mechanisms of endocrine resistance are multifaceted and are likely to evolve over time following various single or combination therapies. The purpose of this review article is to provide general understanding of molecular basis of endocrine resistance of breast cancer and to offer comprehensive review on current treatment options and potential new treatment strategies for endocrine resistant breast cancers. Last but not the least, we discuss current challenges and future directions for management of endocrine resistant breast cancers.

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.

Combination of palbociclib with enzalutamide shows in vitro activity in RB proficient and androgen receptor positive triple negative breast cancer cells

Objectives Triple negative breast cancer (TNBC) lacks specific drug targets and remains challenging. Palbociclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor is approved for metastatic estrogen receptor (ER)-positive and human epithermal growth factor 2 (HER2)-negative breast cancer. The nature of cell cycle inhibition by palbociclib suggests its potential in TNBC cells. Retinoblastoma (RB, a known substrate of CDK4/6) pathway deregulation is a frequent occurrence in TNBC and studies have revealed that pharmacological CDK4/6 inhibition induces a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models. In addition, recent studies reported that anti-androgen therapy shows preclinical efficacy in androgen-receptor (AR)-positive TNBC cells. Here we examined the effect of palbociclib in combination with an anti-androgen enzalutamide in TNBC cells. Method MDA-MB-453, BT-549, MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Protein expressions were assessed by Western blot analysis. Cytostatic effect was examined by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. Results Palbociclib showed inhibitory effect in RB-proficient TNBC cells, and enzalutamide inhibited cell viability in AR-positive TNBC cells. Enzalutamide treatment could enhance the palbociclib-induced cytostatic effect in AR-positive/RB-proficient TNBC cells. In addition, palbociclib-mediated G1 arrest in AR-positive/RB-proficient TNBC cells was attenuated by RB knockdown. Conclusion Our study provided a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists.

Abstract 2087: Pan-HER inhibitor, varlitinib, disrupts HER/ERK signaling and causes apoptosis in triple-negative breast cancer cells

Background: Triple-negative breast cancer (TNBC), characterized by aggressive behavior and poor prognosis, represents an important clinical challenge because there is no well-established target therapy. Therefore, the identification and validation of a targeted therapy for TNBC is an urgent need. Molecular profiling studies have shown some TNBC tumors harboring aberrant epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor (HER) signaling, suggesting therapeutic potential with EGFR inhibitors. Varlitinib (ASLAN001) is a small molecule reversible pan-HER inhibitor of EGFR (HER1), HER2 and HER4. To-date, varlitinib has been extensively investigated in several tumor types, including HER2 positive metastatic breast cancer. TNBC is known to demonstrate expression of EGFR. Since varlitinib also targets EGFR signaling, we hypothesized that it may also have antitumor efficacy in TNBC. Methods: MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptotic effects were examined by flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western blot. Results: We first examined the protein expression of EGFR in a panel of TNBC cell lines. We identified MDA-MB-468 and MDA-MB-231 as EGFR-expressing cell lines. We found that varlitinib significantly inhibited cell viability and induced cell apoptosis in MDA-MB-468 cells but not in MDA-MB-231 cells. MDA-MB-231 cells carry the KRAS G13D mutation that drives downstream ERK signaling. We therefore examined the downstream signaling proteins of EGFR, including PI3K/Akt and MAPK/ERK signaling. Results showed that the protein levels of p-MEK and p-ERK were decreased in varlitinib-sensitive MDA-MB-468 cell lines, but there was no significant change in these phospho-proteins in varlitinib-resistant MDA-MB-231 cells. Furthermore, we found that ERK inhibition resensitized varlitinib-resistant cells to varlitinib-induced cell death. In addition, ectopic expression of ERK reduced the varlitinib-induced apoptosis on varlitinib-sensitive MDA-MB-468 cells. In addition, MDA-MB-468 cells are known to harbor p53-R273H gain-of-function mutation that may activate EGFR-signaling, whether the mutation is associated with varlitinib sensitivity needs further investigation. Conclusions: In this study, we identified TNBC as another tumor type that may be sensitive to varlitinib’s antitumor activity through the inhibition of HER/MAPK signaling and subsequent increase in apoptotic activity. Citation Format: Chun-Yu Liu, Tzu-Ting Huang, Chun-Teng Huang, Hsiu-Ping Yang, Ling-Ming Tseng, Chung-Wai Shiau, Kuen-Feng Chen. Pan-HER inhibitor, varlitinib, disrupts HER/ERK signaling and causes apoptosis in triple-negative breast cancer cells [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 2087. doi:10.1158/1538-7445.AM2017-2087

ER stress‐related ATF6 upregulates CIP2A and contributes to poor prognosis of colon cancer

Endoplasmic reticulum (ER) stress is an adaptive response to various stress conditions and plays emerging roles in cancer. Activating transcription factor 6 (ATF6), one of the three major ER stress transducers, has been shown to contribute to chemoresistance by altering cancer cell survival. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogene, and its expression has been correlated with the prognosis of patients with cancer. In this study, we aimed to explore the relationship between ER stress‐related ATF signaling and CIP2A. We found that CIP2A expression was positively correlated with ATF6 expression by analyzing publicly available RNA sequence data of patients with colorectal cancer (The Cancer Genome Atlas, TCGA). In addition, we demonstrated that tunicamycin‐induced ER stress in vitro upregulated ATF6 and CIP2A. Mechanistically, we found that ATF6 directly bound to the CIP2A promoter and induced CIP2A gene expression, which contributed to colon cancer cell survival. Furthermore, knockdown of CIP2A reduced the viability of cells under ER stress. Most importantly, immunohistochemical analysis of a tissue microarray from a colon cancer patient cohort showed that higher expression levels of ATF6 and CIP2A were associated with a trend toward poor prognosis. Taken together, our results show that ER stress‐related ATF6 upregulates CIP2A and contributes to the prognosis of colon cancer. Targeting CIP2A may disrupt ER stress‐mediated colon cancer cell survival and thus improve the prognosis of patients with colon cancer.

Serine/threonine protein phosphatase 5 is a potential therapeutic target in cholangiocarcinoma

Few molecules are currently verified to be actionable drug targets in cholangiocarcinoma (CCA). Serine/threonine protein phosphatase 5 (PP5) dysregulation is related to several malignancies. However, the role of PP5 in CCA is poorly defined.

The tyrosine kinase inhibitor nintedanib activates SHP-1 and induces apoptosis in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) remains difficult to treat and urgently needs new therapeutic options. Nintedanib, a multikinase inhibitor, has exhibited efficacy in early clinical trials for HER2-negative breast cancer. In this study, we examined a new molecular mechanism of nintedanib in TNBC. The results demonstrated that nintedanib enhanced TNBC cell apoptosis, which was accompanied by a reduction of p-STAT3 and its downstream proteins. STAT3 overexpression suppressed nintedanib-mediated apoptosis and further increased the activity of purified SHP-1 protein. Moreover, treatment with either a specific inhibitor of SHP-1 or SHP-1-targeted siRNA reduced the apoptotic effects of nintedanib, which validates the role of SHP-1 in nintedanib-mediated apoptosis. Furthermore, nintedanib-induced apoptosis was attenuated in TNBC cells expressing SHP-1 mutants with constantly open conformations, suggesting that the autoinhibitory mechanism of SHP-1 attenuated the effects of nintedanib. Importantly, nintedanib significantly inhibited tumor growth via the SHP-1/p-STAT3 pathway. Clinically, SHP-1 levels were downregulated, whereas p-STAT3 was upregulated in tumor tissues, and SHP-1 transcripts were associated with improved disease-free survival in TNBC patients. Our findings revealed that nintedanib induces TNBC apoptosis by acting as a SHP-1 agonist, suggesting that targeting STAT3 by enhancing SHP-1 expression could be a viable therapeutic strategy against TNBC.

Abstract 4192: Targeting SHP-1/p-Lyn signaling shows therapeutic potential in diffuse large B-cell lymphoma

Background: Diffuse large B cell lymphoma (DLBCL) is one aggressive form of non-Hodgkin’s lymphoma. Genetic analyses revealed molecular heterogeneity of DLBCL tumors, classifying the cell-of-origin into two distinct molecular subtypes: germinal center B-cell (GCB) and activated B-cell (ABC). ABC-type DLBCL has a worse survival after upfront chemotherapy compared to GCB-type DLBCL, thus ABC-type DLBCL patients have an unmet medical need that warrants additional research efforts and new therapeutic options. In current study we explored the biological role and potential therapeutic implication of a protein phosphatase SHP-1 in DLBCL. Methods: DLBCL cell lines including ABC-like cell lines U2932, Ly-3 and GC-like cell lines DHL-6, Ly-7 and DB were used for in vitro studies. Cell viability was examined by MTT assay. Apoptotic effects were examined by flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western blot. In vivo therapeutic testing of SHP-1 agonists were performed in nude mice with DLBCL xenografts. Results: We first examined the protein expression of SHP-1 and its downstream p-STAT3 in a panel of DLBCL cell lines, and identified in general SHP-1/p-STAT3 expression was higher in ABC-like cells. Interestingly, the expressions of p-Lyn (Tyr396), p-BTK (Tyr223), key members of B-cell receptor (BCR) signaling pathway, were also higher in ABC-like cells. Knockdown or overexpression of SHP-1 protein expression revealed a reciprocal change of p-Lyn, suggesting SHP-1 negatively regulates phosphorylation of Lyn kinase. Immunoprecipitation experiments confirmed SHP-1 interact with Lyn in DLBCL cells. We previously developed direct SHP-1 agonists, namely SC-43 and SC-60, which could increase SHP-1 activities and induce apoptosis. Here we tested SC-43 and SC-60 in comparison to ibrutinib, a selective Bruton9s tyrosine kinase (BTK) inhibitor. The SHP-1 agonists showed in general superior anti-proliferative and apoptotic effects, comparing to ibrutinib. Mechanistically, SHP-1 agonists enhanced SHP-1 activity, decreased BCR signaling p-Lyn and p-BTK, which led to apoptosis. In addition, SHP-1 agonists also down-regulated p-STAT3 as previously reported, which also contributes to anti-cancer effects. In vivo, SC-43 at doses of 10mg/kg/day and 30mg/kg/day orally showed comparable anti-tumor effects with ibrutinib at doses of 12.5 mg/kg/day and 25 mg/kg/day in mice bearing U2932 xenografts, respectively. Western blot confirmed SC-43 downregulation on p-Lyn and p-BTK in vivo. Conclusions: SHP-1 negatively regulates phosphorylation of Lyn, and targeting SHP-1/p-Lyn with direct SHP-1 agonists shows therapeutic potential in DLBCL. Citation Format: Chun-Yu Liu, Man-Hsin Hung, Ming-Hsien Tsai, Pei-Yi Chu, Tzu-Ting Huang, Chun-Teng Huang, Chung-Wai Shiau, Kuen-Feng Chen. Targeting SHP-1/p-Lyn signaling shows therapeutic potential in diffuse large B-cell lymphoma [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 4192. doi:10.1158/1538-7445.AM2017-4192