Bo Zhai

Inhibition of Akt reverses the acquired resistance to sorafenib by switching protective autophagy to autophagic cell death in hepatocellular carcinoma

Sorafenib is the standard first-line systemic drug for advanced hepatocellular carcinoma (HCC), but the acquired resistance to sorafenib results in limited benefits. Activation of Akt is thought to be responsible for mediating the acquired resistance to sorafenib. The present study aims to examine the underlying mechanism and seek potential strategies to reverse this resistance. Two sorafenib-resistant HCC cell lines, which had been established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition and apoptosis in vitro and in vivo. Sustained exposure to sorafenib activated Akt via the feedback loop of mTOR but independent of protein phosphatase 2A in HCC cells. Autophagy participated in the resistance to sorafenib as inhibition of autophagy reduced the sensitivity of sorafenib-resistant HCC cells to sorafenib, whereas activation of autophagy by rapamycin had the opposite effect. However, rapamycin did not show a synergistic effect with sorafenib to inhibit cell proliferation, while it also activated Akt via a feedback mechanism in sorafenib-resistant HCC cells. Inhibition of Akt reversed the acquired resistance to sorafenib by switching autophagy from a cytoprotective role to a death-promoting mechanism in the sorafenib-resistant HCC cells. Akt inhibition by GDC0068 synergized with sorafenib to suppress the growth of sorafenib-resistant HCC tumors that possessed the sorafenib-resistant feature in vivo. The results have provided evidence for clinical investigation of GDC0068, a novel ATP-competitive pan-Akt inhibitor, as the second-line treatment after the failure of sorafenib-medicated molecular targeted therapy for advanced HCC. Mol Cancer Ther; 13(6); 1589–98. ©2014 AACR.

STAT3 interacts with Skp2/p27/p21 pathway to regulate the motility and invasion of gastric cancer cells.

The interleukin-6 (IL-6)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway mediates cell proliferation and migration. S-phase kinase-associated protein-2 (Skp2) catalyzes the ubiquitylation of p27 and p21. Here we investigated that the cross-talk of the two pathways regulates motility and invasion of gastric cancer SGC7901 and MGC803 cells. Both cell lines endogenously secret IL-6, and blockage of IL-6 or JAK2 inhibited the activation of JAK2 and STAT3. Depletion of STAT3 downregulated Skp2 expression, and thereby increased the expression of p27 and p21. The depletion of STAT3 inhibited the ability of cells to migrate and invade, and impaired the cellular cytoskeleton mainly microtubules; while the depletion of p27 partially restored the impaired ability to migrate, and reversed the impaired microfilaments, further inhibited the ability to invade, but had little effect on microtubules and cellular adhering ability of STAT3-depleted cells. STAT3 depletion inhibited the activity of RhoA and the interaction with stathmin, downregulated the expression of pFAK (phosphorylated focal adhesion kinase), acetylated-tubulin, RECK (reversion-inducing-cysteine-rich protein with kazal motifs) and Sp1, upregulated E-cadherin, and reduced the activities of MMP (matrix metalloproteinase)-2 and -9. The depletion of p27 increased RhoA (Ras homolog family member A) activity, upregulated RECK, and downregulated E-cadherin and Sp1 in STAT3-depleted cells. The results indicate that the interaction between STAT3 and Skp2/p27/p21 pathway plays an important role in mediating the motility, migration and invasion of gastric cancer cells, and inhibition of STAT3 may be a useful therapeutic approach for metastasis of gastric cancer, but caution needs to be taken for its effects on Skp2/p27/p21 pathway.

MiR-21 mediates sorafenib resistance of hepatocellular carcinoma cells by inhibiting autophagy via the PTEN/Akt pathway

Sorafenib resistance remains a major obstacle for the effective treatments of hepatocellular carcinoma (HCC). Recent studies indicate that activated Akt contributes to the acquired resistance to sorafenib, and miR-21 dysregulates phosphatase and tensin homolog (PTEN), which inhibits Akt activation. Sorafenib-resistant HCC cells were shown to be refractory to sorafenib-induced growth inhibition and apoptosis. Akt and its downstream factors were highly activated and/or upregulated in sorafenib-resistant cells. Inhibition of autophagy decreased the sensitivity of sorafenib-resistant cells to sorafenib, while its induction had the opposite effect. Differential screening of miRNAs showed higher levels of miR-21 in sorafenib-resistant HCC cells. Exposure of HCC cells to sorafenib led to an increase in miR-21 expression, a decrease in PTEN expression and sequential Akt activation. Transfection of miR-21 mimics in HCC cells restored sorafenib resistance by inhibiting autophagy. Anti-miR-21 oligonucleotides re-sensitized sorafenib-resistant cells by promoting autophagy. Inhibition of miR-21 enhances the efficacy of sorafenib in treating sorafenib-resistant HCC tumors in vivo. We conclude that miR-21 participates in the acquired resistance of sorafenib by suppresing autophagy through the Akt/PTEN pathway. MiR-21 could serve as a therapeutic target for overcoming sorafenib resistance in the treatment of HCC.

Upregulation of HIF-2α induced by sorafenib contributes to the resistance by activating the TGF-α/EGFR pathway in hepatocellular carcinoma cells.

Sorafenib, the first-line systemic drug for advanced hepatocellular carcinoma (HCC), has demonstrated limited benefits with very low response rates. Thus it is essential to investigate the underlying mechanisms for the resistance to sorafenib and seek potential strategy to enhance its efficacy. Hypoxic cells inside solid tumors are extremely resistant to therapies as their survival ability is increased due to the cellular adaptive response to hypoxia, which is controlled by hypoxia-inducible factor (HIF)-1 and HIF-2. Sorafenib inhibits HIF-1α synthesis, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways and providing a mechanism for more aggressive growth of tumors. The present study has demonstrated that upregulation of HIF-2α induced by sorafenib contributes to the resistance of hypoxic HCC cells by activating the transforming growth factor (TGF)-α/epidermal growth factor receptor (EGFR) pathway. Blocking the TGF-α/EGFR pathway by gefitinib, a specific EGFR inhibitor, reduced the activation of STAT (signal transducer and activator of transcription) 3, AKT and ERK (extracellular signal-regulated kinase), and synergized with sorafenib to inhibit proliferation and induce apoptosis of hypoxic HCC cells. Transfection of HIF-2α siRNA into HCC cells downregulated the expression of VEGF (vascular endothelial growth factor), cyclin D1, HIF-2α and TGF-α, and inhibited the activation of EGFR. HIF-2α siRNA inhibited the proliferation and promoted the apoptosis of HCC cells in vitro, and synergized with sorafenib to suppress the growth of HCC tumors in vivo. The results indicate that targeting HIF-2α-mediated activation of the TGF-α/EGFR pathway warrants further investigation as a potential strategy to enhance the efficacy of sorafenib for treating HCC.

Up-regulation of survivin by AKT and hypoxia-inducible factor 1α contributes to cisplatin resistance in gastric cancer

This study investigated the contribution of survivin and its upstream regulators, AKT and hypoxia‐inducible factor 1α (HIF–1α), to the resistance of gastric cancer cells to cisplatin (CDDP). We found that over‐expression of survivin increased the resistance of SGC7901 and BGC823 gastric cancer cells to CDDP. Its over‐expression abrogated CDDP‐induced inhibition of cell proliferation and CDDP‐induced cell apoptosis. In contrast, down‐regulation of survivin expression using small hairpin RNA (shRNA) vectors and the small‐molecule inhibitor YM155, or inhibition of survivin function using a recombinant cell‐permeable dominant‐negative survivin protein (dNSur9), promoted CDDP‐induced apoptosis. CDDP‐resistant sub‐lines generated from the parental SGC7901 and BGC823 cells by exposure to increasing concentrations of CDDP expressed higher levels of HIF–1α and survivin in response to hypoxia, and higher levels of phosphorylated AKT (pAKT). Specific inhibition of AKT reduced the expression of HIF–1α and survivin, whereas specific inhibition or depletion of HIF–1α reduced survivin expression but had no effect on the expression of phosphorylated AKT. The expression levels of survivin affected the therapeutic efficacy of CDDP in treating gastric tumors in mice. Specific inhibition of survivin, AKT and HIF–1α enhanced the sensitivity of CDDP‐resistant cells to CDDP. Specific inhibition of survivin, AKT and HIF‐1α synergized with CDDP to suppress the growth of gastric tumors that had been engineered to overexpress survivin. In summary, the results provide evidence that up‐regulation of survivin by AKT and HIF–1α contributes to CDDP resistance, indicating that inhibition of these pathways may be a potential strategy for overcoming CDDP resistance in the treatment of gastric cancer.

Downregulation of Skp2 inhibits the growth and metastasis of gastric cancer cells in vitro and in vivo

S-phase kinase-associated protein-2 (Skp2) is overexpressed in human cancers and associated with poor prognosis. Skp2 acts as an oncogenic protein by enhancing cancer cell growth and tumor metastasis. The present study has demonstrated that small hairpin RNA (shRNA)-mediated downregulation of Skp2 markedly inhibits the viability, proliferation, colony formation, migration, invasion, and apoptosis of human gastric cancer MGC803 cells, which express a high level of Skp2. In contrast, Skp2 shRNA had only a slight effect on the above properties of BGC823 cells, which express a low level of Skp2. In accord, knockdown of Skp2 suppressed the ability of MGC803 cells to form tumors and to metastasize to the lungs of mice, and the growth of established tumors, by inhibiting cell proliferation and enhancing cell apoptosis. In contrast, overexpression of Skp2 promoted tumorigenesis of BGC823 cells in mice. Skp2 depletion induced cell cycle arrest in the G1/S phase by upregulating p27, p21, and p57 and downregulating cyclin E and cyclin-dependent kinase 2. Skp2 depletion also increased caspase-3 activity, impeded the ability of cells to form filopoidia and locomote, upregulated RECK (reversion-inducing cysteine-rich protein with kazal motifs), and downregulated matrix metalloproteinase (MMP)-2 and MMP-9 activity and expression. The results suggest that downregulating Skp2 warrants investigation as a promising strategy to treat gastric cancers that express high levels of Skp2.

Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation.

Bufalin extracts are a part of traditional Chinese medicine, Chansu. In the current study, we investigated the effect of bufalin on the proliferation of the human hepatocellular carcinoma (HCC) cell lines, Huh-7 and HepG-2, and explored the therapeutic potential of the drug. Our results demonstrated that bufalin markedly inhibited cell proliferation and promoted apoptosis in the Huh-7 and HepG-2 cells in vitro. The underlying mechanism of the bufalin-induced apoptosis was the induction of endoplasmic reticulum (ER) stress via the IRE1–JNK pathway. In addition, during the ER stress response, the autophagy pathway, characterized by the conversion of LC3-I to LC3-II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression and stimulation of autophagic flux. Our data supported the pro-survival role of bufalin-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors; the involvement of the IRE1 pathway in the ER stress-induced autophagy was also demonstrated when the expression of IRE1 and CHOP was silenced using siRNA. These data indicate that combining bufalin with a specific autophagy inhibitor could be a promising therapeutic approach for the treatment of HCC.

Secretory clusterin contributes to oxaliplatin resistance by activating Akt pathway in hepatocellular carcinoma.

Secretory clusterin (sCLU) is expressed in numerous cancers and is associated with the resistance to chemotherapy. However, the role of sCLU in the resistance of hepatocellular carcinoma (HCC) to oxaliplatin (OXA), a recently used third‐generation platinum agent, remains unclear. The stable transfectants that are depleted of or overexpress sCLU and OXA‐resistant cells were generated using human HCC cells. Overexpression of sCLU abrogated OXA‐induced inhibition of cell growth and cell apoptosis, but depletion of sCLU synergized with OXA to inhibit cell growth and enhance cell apoptosis, by regulating proteins involved in mitochondrial apoptosis pathways, such as Bcl‐2, Bax, Bcl‐xL and caspase‐9, and affecting phosphorylation of Akt and GSK‐3β. Overexpression of sCLU in either OXA‐resistant cells or stable transfectants that overexpress sCLU significantly increased phosphorylated Akt. However, specific inhibition of Akt enhanced sensitivity of sCLU‐overexpressing cells to OXA, but had no effect on sCLU expression, suggesting that the regulatory effects between sCLU and pAkt may be in a one‐way manner in HCC cells. The expression levels of sCLU affected the therapeutic efficacy of OXA to treat HCC tumors established in immunodeficiency mice. The results have demonstrated that sCLU contributes to OXA resistance by activating Akt pathway, indicating that sCLU may be a novel molecular target for overcoming OXA resistance in HCC.

Arsenic trioxide potentiates the anti-cancer activities of sorafenib against hepatocellular carcinoma by inhibiting Akt activation

Sorafenib is the standard first-line systemic drug for advanced hepatocellular carcinoma (HCC), but it also induces the activation of Akt, which contributes to the mechanisms for the resistance to sorafenib. Arsenic trioxide (ATO) is a currently clinically used anticancer drug and displays its anticancer activities by inhibiting Akt activation. Therefore, we hypothesized that ATO may potentiate the anti-cancer activities of sorafenib against HCC. The results have demonstrated that ATO synergized with sorafenib to inhibit the proliferation and promote the apoptosis of HCC cells by diminishing the increased activation of Akt by sorafenib. ATO was shown to inhibit the expression or activation of Akt downstream factors, including glycogen synthase kinase (GSK)-3β, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (S6K), and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which regulate cell apoptosis and were upregulated or activated by sorafenib. Both sorafenib and ATO downregulated the expression of cyclin D1, resulting in HCC cells arrested at G0/G1 phase. ATO downregulated the expression of Bcl-2 and Bcl-xL and upregulated the expression of Bax, indicating that ATO could induce the apoptosis of HCC cells through the intrinsic pathways; but sorafenib showed little effects on these proteins of Bcl-2 family. ATO synergized with sorafenib to suppress the growth of HCC tumors established in mice by inhibiting the proliferation and inducing the apoptosis of HCC cells in situ. These results indicate that ATO may be a potential agent that given in combination with sorafenib acts synergistically for treating HCC.

Meloxicam Executes Its Antitumor Effects against Hepatocellular Carcinoma in COX-2- Dependent and -Independent Pathways

Background Cyclooxygenase (COX)-2 is overexpressed in many types of cancers including hepatocellular carcinoma (HCC). Meloxicam, a selective COX-2 inhibitor, has shown potential therapeutic effects against HCC, but the mechanisms accounting for its anti-cancer activities remain unclear. Methods and Findings Meloxicam inhibited the ability of human HCC cells expressing higher levels of COX-2 to migrate, invade, adhere and form colonies through upregulating the expression of E-cadherin and downregulating the expression of matrix metalloproteinase (MMP) -2. Meloxicam induced cell apoptosis by upregulating pro-apoptotic proteins including Bax and Fas-L, and downregulating anti-apoptotic proteins including survivin and myeloid cell leukemia-1 (Mcl-1), through inhibiting phosphorylation of AKT. Addition of prostaglandin E2 (PGE2), the major product of COX-2, could abrogate the effects of meloxicam on the expression of survivin and myeloid cell leukemia-1 (Mcl-1), but not Bax and Fas-L, indicating that meloxicam induces cell apoptosis via both COX-2-dependent and -independent pathways. Meloxicam also induced cell autophagy by upregulating Beclin 1 and light chain 3-II. Specific inhibition of autophagy by 3-methyladenine and chloroquine had little effect on cell apoptosis but could enhance the pro-apoptotic effects of meloxicam by further upregulating the expression of Bax. Conclusions Meloxicam executes its antitumor effects by targeting the COX-2/MMP-2/E-cadherin, AKT, apoptotic and autophagic pathways in COX-2-dependent and -independent pathways, and inhibition of cell autophagy could help to overcome the resistance to meloxicam-induced apoptosis in HCC.