Chun-Teng Huang

Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A–dependent phospho-Akt inactivation in estrogen receptor–negative human breast cancer cells

IntroductionTamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism.MethodsIn total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice.ResultsTamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors.ConclusionsInhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel “off-target“ mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.

Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells

We tested the efficacy of lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and epidermal growth factor receptor (EGFR) pathways, in a panel of triple-negative breast cancer (TNBC) cells, and examined the drug mechanism. Lapatinib showed an anti-proliferative effect in HCC 1937, MDA-MB-468, and MDA-MB-231 cell lines. Lapatinib induced significant apoptosis and inhibited CIP2A and p-Akt in a dose and time-dependent manner in the three TNBC cell lines. Overexpression of CIP2A reduced lapatinib-induced apoptosis in MDA-MB-468 cells. In addition, lapatinib increased PP2A activity (in relation to CIP2A inhibition). Moreover, lapatinib-induced apoptosis and p-Akt downregulation was attenuated by PP2A antagonist okadaic acid. Furthermore, lapatinib indirectly decreased CIP2A transcription by disturbing the binding of Elk1 to the CIP2A promoter. Importantly, lapatinib showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and suppressed CIP2A as well as p-Akt in these xenografted tumors. In summary, inhibition of CIP2A determines the effects of lapatinib-induced apoptosis in TNBC cells. In addition to being a dual tyrosine kinase inhibitor of HER2 and EGFR, lapatinib also inhibits CIP2A/PP2A/p-Akt signaling in TNBC cells.

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

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.

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

Abstract 4201: Combination of palbociclib with enzalutamide shows in vitro activity in RB-proficient and androgen receptor-positive triple-negative breast cancer cells

Background Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have in vitro synergistic effects and significant clinical efficacy in combination with hormone therapy for estrogen receptor (ER)-positive advanced breast cancer. However, the effects of CDK4/6 inhibitors in triple negative breast cancer (TNBC) are not well-elucidated. Retinoblastoma (RB, a known substrate of CDK4/6) protein pathway deregulation is a frequent occurrence in TNBC and in vitro studies have revealed that pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models, suggesting that RB may be a biomarker for selecting patients receiving CDK4/6 inhibitors. In addition, anti-androgen therapy have been shown preclinical efficacy in androgen-receptor (AR) positive TNBC cells. Here we tested the combination effect of a CDK4/6 inhibitor palbociclib with an AR antagonist enzalutamide in TNBC cells. Methods MDA-MB-231, MDA-MB-468 and MDA-MB-453 TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptotic effects were examined by flow cytometry. Signal transduction pathways in cells were assessed by Western blot. Results We correlated the expressions of AR and pRB status in these TNBC cells and identified that MDA-MB-453 were AR-positive/pRB-positive, MDA-MB-231 were AR-negative/pRB-positive, and MDA-MB-468 were AR-negative/pRB-negative cells. Since CDK4/6 inhibition leads to dephosphorylation of pRB, we examined effects of palbociclib and found that palbociclib significantly inhibited the cell growth of pRB-positive cell lines MDA-MB-453 and MDA-MB-231, but did not affect pRB-negative MDA-MB-468 cells. Further combining palbociclib with enzalutamide showed that the growth inhibitory effects of combination therapy were additive in AR-positive/pRB-positive MDA-MB-453 cells, but not in AR-negative/pRB-positive MDA-MB-231 cells, nor AR/pRB double negative MDA-MB-468 cells. Moreover, palbociclib-induced G1/S arrest only exhibited in pRB-proficient MDA-MB-453 and MDA-MB-231 cells. In contrast, enzalutamide did not affect the cell cycle on any of these TNBC cell lines but inhibited phosphorylation of AR signaling in AR-positive MDA-MB-453 cells. In addition, both palbociclib and enzalutamide induced little apoptosis, suggesting cytostatic effects of these agents. Conclusions In summary, both pRB and AR status are important for response of TNBC cells to palbociclib. Combination of palbociclib with enzalutamide shows more prominent cytostatic effects in RB proficient and AR positive TNBC cells, comparing to AR-negative/pRB-positive or AR/pRB double negative TNBC cells. Our data provide a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists. Citation Format: Ling-Ming Tseng, Tzu-Ting Huang, Chun-Teng Huang, Ka-Yi Lau, Chun-Yu Liu, Chung-Wai Shiau, Kuen-Feng Chen. Combination of palbociclib with enzalutamide shows in vitro activity in RB-proficient and androgen receptor-positive 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 4201. doi:10.1158/1538-7445.AM2017-4201

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.