Ming-Hsien Tsai

A combination of sorafenib and SC-43 is a synergistic SHP-1 agonist duo to advance hepatocellular carcinoma therapy.

Sorafenib is the first and currently the only standard treatment for advanced hepatocellular carcinoma (HCC). We previously developed a sorafenib derivative SC-43, which exhibits much more enhanced anti-HCC activity than sorafenib and also promotes apoptosis in sorafenib-resistant HCC cells. Herein, a novel sorafenib plus combination therapy was developed by coupling sorafenib treatment with SC-43. Both sorafenib and SC-43 are proven Src homology region 2 domain containing phosphatase 1 (SHP-1) agonists. The combined actions of sorafenib and SC-43 enhanced SHP-1 activity, which was associated with diminished STAT3-related signals and stronger expression of apoptotic genes above that of either drug alone, culminating in increased cell death. Decreased p-STAT3 signaling and tumor size, as well as increased SHP-1 activity were observed in mice receiving the combination therapy in a subcutaneous HCC model. More reduced orthotopic HCC tumor size and prolonged survival were also observed in mice in the combination treatment arm compared to mice in either of the monotherapy arms. These results in the preclinical setting pave the way for further clinical studies to treat unresectable HCC.

Protein tyrosine phosphatase 1B dephosphorylates PITX1 and regulates p120RasGAP in hepatocellular carcinoma

The effective therapeutic targets for hepatocellular carcinoma remain limited. Pituitary homeobox 1 (PITX1) functions as a tumor suppressor in hepatocarcinogenesis by regulating the expression level of Ras guanosine triphosphatase‐activating protein. Here, we report that protein tyrosine phosphatases 1B (PTP1B) directly dephosphorylated PITX1 at Y160, Y175, and Y179 to further weaken the protein stability of PITX. The PTP1B‐dependent decline of PITX1 reduced its transcriptional activity for p120RasGAP (RASA1), a Ras guanosine triphosphatase‐activating protein. Both silencing of PTP1B and PTP1B inhibitor up‐regulated the PITX1‐p120RasGAP axis through hyperphosphorylation of PITX1. Sorafenib, the first and only targeted drug approved for hepatocellular carcinoma, directly decreased PTP1B activity and promoted the expression of PITX1 and p120RasGAP by PITX1 hyperphosphorylation. Molecular docking also supported the potential interaction between PTP1B and sorafenib. PTP1B overexpression impaired the sensitivity of sorafenib in vitro and in vivo, implying that PTP1B has a significant effect on sorafenib‐induced apoptosis. In sorafenib‐treated tumor samples, we further found inhibition of PTP1B activity and up‐regulation of the PITX1‐p120RasGAP axis, suggesting that PTP1B inhibitor may be effective for the treatment of hepatocellular carcinoma. By immunohistochemical staining of hepatic tumor tissue from 155 patients, the expression of PTP1B was significantly in tumor parts higher than nontumor parts (P = 0.02). Furthermore, high expression of PTP1B was significantly associated with poor tumor differentiation (P = 0.031). Conclusion: PTP1B dephosphorylates PITX1 to weaken its protein stability and the transcriptional activity for p120RasGAP gene expression and acts as a determinant of the sorafenib‐mediated drug effect; targeting the PITX1‐p120RasGAP axis with a PTP1B inhibitor may provide a new therapy for patients with hepatocellular carcinoma. (Hepatology 2016;63:1528‐1543)

Palbociclib induces activation of AMPK and inhibits hepatocellular carcinoma in a CDK4/6 independent manner

Palbociclib, a CDK4/6 inhibitor, has recently been approved for hormone receptor‐positive breast cancer patients. The effects of palbociclib as a treatment for other malignancies, including hepatocellular carcinoma (HCC), are of great clinical interest and are under active investigation. Here, we report the effects and a novel mechanism of action of palbociclib in HCC. We found that palbociclib induced both autophagy and apoptosis in HCC cells through a mechanism involving 5′ AMP‐activated protein kinase (AMPK) activation and protein phosphatase 5 (PP5) inhibition. Blockade of AMPK signals or ectopic expression of PP5 counteracted the effect of palbociclib, confirming the involvement of the PP5/AMPK axis in palbociclib‐mediated HCC cell death. However, CDK4/6 inhibition by lentivirus‐mediated shRNA expression did not reproduce the effect of palbociclib‐treated cells, suggesting that the anti‐HCC effect of palbociclib is independent of CDK4/6. Moreover, two other CDK4/6 inhibitors (ribociclib and abemaciclib) had minimal effects on HCC cell viability and the PP5/AMPK axis. Palbociclib also demonstrated significant tumor‐suppressive activity in a HCC xenograft model, which was associated with upregulation of pAMPK and PP5 inhibition. Finally, we analyzed 153 HCC clinical samples and found that PP5 expression was highly tumor specific and was associated with poor clinical features. Taken together, we conclude that palbociclib exerted antitumor activity against HCC through the PP5/AMPK axis independent of CDK4/6. Our findings provide a novel mechanistic basis for palbociclib and reveal the therapeutic potential of targeting PP5/AMPK signaling with a PP5 inhibitor for the treatment of hepatocellular carcinoma.

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase

ABSTRACT The serine‐threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress‐induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral‐mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP‐activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC.

SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma

Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

Inhibition of protein phosphatase 5 suppresses non-small cell lung cancer through AMP-activated kinase activation

OBJECTIVESnNon-small cell lung cancer (NSCLC) continues to be the top cause of cancer death. To improve the treatment of lung cancer, there is necessity to identify novel oncogenes and investigate their effects on lung carcinogenesis. Protein phosphatase 5 (PP5) has long been known to regulate stress-induced apoptosis and cell proliferation. Recently, PP5 has been found overexpressed and emerged as a viable therapeutic target in various human cancers, but its role in NSCLC remains elusive.nnnMATERIALS AND METHODSnThe expression of PP5 in NSCLC cell lines (A549, H358, and H460) and human tumor samples were examined. Protein phosphatase inhibitors, cantharidin and norcantharidin, were used as proof-of-concept compounds to investigate the pathological function of PP5 in NSCLC. Apoptosis and cellular signaling were analyzed. In vivo efficacy was determined in nude mice with H460 xenograft.nnnRESULTS AND CONCLUSIONnWe found that PP5 was more highly expressed in human lung tumor samples than in adjacent normal tissues. Overexpression of PP5 promoted cell proliferation, colony formation, and sphere-forming ability of A549 cells. Inhibition of PP5 phosphatase activity by cantharidin induced significant apoptosis and upregulated AMP-activated protein kinase (AMPK) signaling. Importantly, we found that PP5-mediated dephosphorylation of AMPK determines the in vitro anti-NSCLC effects of cantharidin. Consistent with our in vitro data, PP5 inhibition suppressed H460 tumor growth and upregulated p-AMPK in tumor samples. Our results demonstrate that PP5 inhibition suppresses tumor growth via activating AMPK signaling. Targeting oncogenic PP5 represents an attractive therapeutic strategy for treating lung 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.

Antagonizing SET Augments the Effects of Radiation Therapy in Hepatocellular Carcinoma through Reactivation of PP2A-Mediated Akt Downregulation

Increasing evidence suggests that SET functions as an oncoprotein and promotes cancer survival and therapeutic resistance. However, whether SET affects radiation therapy (RT)-mediated anticancer effects has not yet been explored. We investigated the impact of SET on RT sensitivity in hepatocellular carcinoma (HCC). Using colony and hepatosphere formation assays, we found that RT-induced proliferative inhibition was critically associated with SET expression. We next tested a novel SET antagonist, N4-(3-ethynylphenyl)-6,7-dimethoxy-N2-(4-phenoxyphenyl) quinazoline-2,4-diamine (EMQA), in combination with RT. We showed that additive use of EMQA significantly enhanced the effects of RT against HCC in vitro and in vivo. Notably, compared with mice receiving either RT or EMQA alone, the growth of PLC5 xenografted tumor in mice receiving RT plus EMQA was significantly reduced without compromising treatment tolerability. Furthermore, we proved that antagonizing SET to restore protein phosphatase 2A-mediated phospho-Akt (p-AKT) downregulation was responsible for the synergism between EMQA and RT. Our data demonstrate a new oncogenic property of SET and provide preclinical evidence that combining a SET antagonist and RT may be effective for treatment of HCC. Further investigation is warranted to validate the clinical relevance of this approach.

Palbociclib enhances radiosensitivity of hepatocellular carcinoma and cholangiocarcinoma via inhibiting ataxia telangiectasia–mutated kinase–mediated DNA damage response

AIMnPalbociclib is an oral cyclin-dependent kinase 4/6 inhibitor, which is efficacious in treating breast cancer. Currently, there are numerous active clinical trials testing palbociclib alone or in combination with other medications for treating various types of malignancies. Here, we evaluated the anti-cancer effect of palbociclib in combination with radiation therapy (RT) for treating human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA)xa0and addressed the molecular mechanism behind the combination therapy.nnnMETHODSnImmunofluorescence staining of γH2AX or 53BP1 was used to determine the effect of palbociclib on double-strand break (DSB) repair. Clonogenic assays, sphere formationxa0and cell death ELISA were performed to study the sensitising effect of palbociclib on radiation-induced cytotoxicity. Signal alteration in DSB repair pathways was examined by Western blot analysis. Finally, we evaluated the inxa0vivo anti-cancer activity and the associated molecular events of the combination therapy in a preclinical HCC xenograft model.nnnRESULTSnPalbociclib affected the kinetics of DNA repair and enhanced the radiation sensitivity of HCC and CCA cells. Importantly, we found that palbociclib inhibits ataxia telangiectasia-mutated (ATM) kinase, the key upstream kinase responding to RT-induced DSBs. Furthermore, we showed that the inhibitory effect of palbociclib on RT-induced ATM kinase activation is mediated by protein phosphatase 5 (PP5). Both inxa0vitro and inxa0vivo investigations revealed that the inhibition of the PP5-ATM axis by palbociclib after DNA damage is responsible for the synergism between palbociclib and RT.nnnCONCLUSIONnOur findings provide a novel combination strategy against liver cancer cells. Clinical trials using palbociclib as an adjuvant in RT are warranted.

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