Identification of HMGCR as the anticancer target of physapubenolide against melanoma cells by in silico target prediction.

Physapubenolide (PB), a withanolide-type compound extracted from the traditional herb Physalis minima L., has been demonstrated to exert remarkable cytotoxicity against cancer cells; however, its molecular mechanisms are still unclear. In this study, we demonstrated that PB inhibited cell proliferation and migration in melanoma cells by inducing cell apoptosis. The anticancer activity of PB was further verified in a melanoma xenograft model. To explore the mechanism underlying the anticancer effects of PB, we carried out an in silico target prediction study, which combined three approaches (chemical similarity searching, quantitative structure-activity relationship (QSAR), and molecular docking) to identify the targets of PB, and found that PB likely targets 3-hydroxy-methylglutaryl CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, which promotes cancer cell proliferation, migration, and metastasis. We further demonstrated that PB interacted with HMGCR, decreased its protein expression and inhibited the HMGCR/YAP pathway in melanoma cells. In addition, we found that PB could restore vemurafenib sensitivity in vemurafenib-resistant A-375 cells, which was correlated with the downregulation of HMGCR. In conclusion, we demonstrate that PB elicits anticancer action and enhances sensitivity to vemurafenib by targeting HMGCR.

Inhibition of AXL enhances chemosensitivity of human ovarian cancer cells to cisplatin via decreasing glycolysis.

Anexelekto (AXL), a member of the TYRO3-AXL-MER (TAM) family of receptor tyrosine kinases (RTK), is overexpressed in varieties of tumor tissues and promotes tumor development by regulating cell proliferation, migration and invasion. In this study, we investigated the role of AXL in regulating glycolysis in human ovarian cancer (OvCa) cells. We showed that the expression of AXL mRNA and protein was significantly higher in OvCa tissue than that in normal ovarian epithelial tissue. In human OvCa cell lines suppression of AXL significantly inhibited cell proliferation, and increased the sensitivity of OvCa cells to cisplatin, which also proved by nude mice tumor formation experiment. KEGG analysis showed that AXL was significantly enriched in the glycolysis pathways of cancer. Changes in AXL expression in OvCa cells affect tumor glycolysis. We demonstrated that the promotion effect of AXL on glycolysis was mediated by phosphorylating the M2 isoform of pyruvate kinase (PKM2) at Y105. AXL expression was significantly higher in cisplatin-resistant OvCa cells A2780/DDP compared with the parental A2780 cells. Inhibition of AXL decreased the level of glycolysis in A2780/DDP cells, and increased the cytotoxicity of cisplatin against A2780/DDP cells, suggesting that AXL-mediated glycolysis was associated with cisplatin resistance in OvCa. In conclusion, this study demonstrates for the first time that AXL is involved in the regulation of the Warburg effect. Our results not only highlight the clinical value of targeting AXL, but also provide theoretical basis for the combination of AXL inhibitor and cisplatin in the treatment of OvCa.

PKM2-c-Myc-Survivin Cascade Regulates the Cell Proliferation, Migration, and Tamoxifen Resistance in Breast Cancer.

The M2 isoform of pyruvate kinase (PKM2), as a key glycolytic enzyme, plays important roles in tumorigenesis and chemotherapeutic drug resistance. However, the intricate mechanism of PKM2 as a protein kinase regulating breast cancer progression and tamoxifen resistance needs to be further clarified. Here, we reported that PKM2 controls the expression of survivin by phosphorylating c-Myc at Ser-62. Functionally, PKM2 knockdown suppressed breast cancer cell proliferation and migration, which could be rescued by overexpression of survivin. Interestingly, we found that the level of PKM2 expression was upregulated in the tamoxifen resistant breast cancer cells MCF-7/TAMR, and knockdown of PKM2 sensitized the cells to 4-hydroxytamoxifen (4OH-T). In addition, the elevated level of PKM2 correlates with poor relapse-free survival in breast cancer patients treated with tamoxifen. Overall, our findings demonstrated that PKM2-c-Myc-survivin cascade regulated the proliferation, migration and tamoxifen resistance of breast cancer cells, suggesting that PKM2 represents a novel prognostic marker and an attractive target for breast cancer therapeutics, and that PKM2 inhibitor combined with tamoxifen may be a promising strategy to reverse tamoxifen resistance in breast cancer patients.

RSK2 protects human breast cancer cells under endoplasmic reticulum stress through activating AMPKα2-mediated autophagy.

Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.

A multi-scale systems pharmacology approach uncovers the anti-cancer molecular mechanism of Ixabepilone.

It has been realized that FDA approved drugs may have more molecular targets than is commonly thought. Thus, to find the exact drug-target interactions (DTIs) is of great significance for exploring the new molecular mechanism of drugs. Here, we developed a multi-scale system pharmacology (MSSP) method for the large-scale prediction of DTIs. We used MSSP to integrate drug-related and target-related data from multiple levels, the network structural data formed by known drug-target relationships for predicting likely unknown DTIs. Prediction results revealed that Ixabepilone, an epothilone B analog for treating breast cancer patients, may target Bcl-2, an oncogene that contributes to tumor progression and therapy resistance by inhibiting apoptosis. Furthermore, we demonstrated that Ixabepilone could bind with Bcl-2 and decrease its protein expression in breast cancer cells. The down-regulation of Bcl-2 by Ixabepilone is resulted from promoting its degradation by affecting p-Bcl-2. We further found that Ixabepilone could induce autophagy by releasing Beclin1 from Beclin1/Bcl-2 complex. Inhibition of autophagy by knockdown of Beclin1 or pharmacological inhibitor augmented apoptosis, thus enhancing the antitumor efficacy of Ixabepilone against breast cancer cells in vitro and in vivo. In addition, Ixabepilone also decreases Bcl-2 protein expression and induces cytoprotective autophagy in human hepatic carcinoma and glioma cells. In conclusion, this study not only provides a feasible and alternative way exploring new molecular mechanisms of drugs by combing computation DTI prediction, but also reveals an effective strategy to reinforce the antitumor efficacy of Ixabepilone.

UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer.

Purpose: To determine the role of UCH-L1 in regulating ERα expression, and to evaluate whether therapeutic targeting of UCH-L1 can enhance the efficacy of anti-estrogen therapy against breast cancer with loss or reduction of ERα. Methods: Expressions of UCH-L1 and ERα were examined in breast cancer cells and patient specimens. The associations between UCH-L1 and ERα, therapeutic response and prognosis in breast cancer patients were analyzed using multiple databases. The molecular pathways by which UCH-L1 regulates ERα were analyzed using immunoblotting, qRT-PCR, immunoprecipitation, ubiquitination, luciferase and ChIP assays. The effects of UCH-L1 inhibition on the efficacy of tamoxifen in ERα (-) breast cancer cells were tested both in vivo and in vitro. Results: UCH-L1 expression was conversely correlated with ERα status in breast cancer, and the negative regulatory effect of UCH-L1 on ERα was mediated by the deubiquitinase-mediated stability of EGFR, which suppresses ERα transcription. High expression of UCH-L1 was associated with poor therapeutic response and prognosis in patients with breast cancer. Up-regulation of ERα caused by UCH-L1 inhibition could significantly enhance the efficacy of tamoxifen and fulvestrant in ERα (-) breast cancer both in vivo and in vitro. Conclusions: Our results reveal an important role of UCH-L1 in modulating ERα status and demonstrate the involvement of UCH-L1-EGFR signaling pathway, suggesting that UCH-L1 may serve as a novel adjuvant target for treatment of hormone therapy-insensitive breast cancers. Targeting UCH-L1 to sensitize ER negative breast cancer to anti-estrogen therapy might represent a new therapeutic strategy that warrants further exploration.

DNA Repair Pathways in Cancer Therapy and Resistance.

DNA repair pathways are triggered to maintain genetic stability and integrity when mammalian cells are exposed to endogenous or exogenous DNA-damaging agents. The deregulation of DNA repair pathways is associated with the initiation and progression of cancer. As the primary anti-cancer therapies, ionizing radiation and chemotherapeutic agents induce cell death by directly or indirectly causing DNA damage, dysregulation of the DNA damage response may contribute to hypersensitivity or resistance of cancer cells to genotoxic agents and targeting DNA repair pathway can increase the tumor sensitivity to cancer therapies. Therefore, targeting DNA repair pathways may be a potential therapeutic approach for cancer treatment. A better understanding of the biology and the regulatory mechanisms of DNA repair pathways has the potential to facilitate the development of inhibitors of nuclear and mitochondria DNA repair pathways for enhancing anticancer effect of DNA damage-based therapy.

Eukaryotic elongation factor-2 kinase regulates the cross-talk between autophagy and pyroptosis in doxorubicin-treated human melanoma cells in vitro.

Eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has been shown to play an important role in modulating autophagy and apoptosis in tumor cells under various stresses. In this study, we investigated the regulatory role of eEF-2K in pyroptosis (a new form of programmed necrosis) in doxorubicin-treated human melanoma cells. We found that doxorubicin (0.5-5 µmol/L) induced pyroptosis in melanoma cell lines SK-MEL-5, SK-MEL-28, and A-375 with high expression of DFNA5, but not in human breast cancer cell line MCF-7 with little expression of DFNA5. On the other hand, doxorubicin treatment activated autophagy in the melanoma cells; inhibition of autophagy by transfecting the cells with siRNA targeting Beclin1 or by pretreatment with chloroquine (20 µmol/L) significantly augmented pyroptosis, thus sensitizing the melanoma cells to doxorubicin. We further demonstrated that doxorubicin treatment activated eEF-2K in the melanoma cells, and silencing of eEF-2K blunted autophagic responses, but promoted doxorubicin-induced pyroptotic cell death. Taken together, the above results demonstrate that eEF-2K dictates the cross-talk between pyroptosis and autophagy in doxorubicin-treated human melanoma cells; suppression of eEF-2K results in inhibiting autophagy and augmenting pyroptosis, thus modulating the sensitivity of melanoma cells to doxorubicin, suggesting that targeting eEF-2K may reinforce the antitumor efficacy of doxorubicin, offering a new insight into tumor chemotherapy.

Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway.

Autophagy, a form of cellular self-digestion by lysosome, is associated with various disease processes including cancers, and modulating autophagy has shown promise in the treatment of various malignancies. A number of natural products display strong antitumor activity, yet their mechanisms of action remain unclear. To gain a better understanding of how traditional Chinese medicine agents exert antitumor effects, we screened 480 natural compounds for their effects on autophagy using a high content screening assay detecting GFP-LC3 puncta in HeLa cells. Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), was identified as a potent activator of autophagy. The activation of autophagy by TBMS1 was evidenced by increased LC3-II amount and GFP-LC3 dots, observation of autophagosomes under electron microscopy, and enhanced autophagic flux. To explore the mechanisms underlying TBMS1-activated autophagy, we performed cheminformatic analyses and surface plasmon resonance (SPR) binding assay that showed a higher likelihood of the binding between Akt protein and TBMS1. In three human breast cancer cell lines, we demonstrated that Akt-mTOR-eEF-2K pathway was involved in TBMS1-induced activation of autophagy, while Akt-mediated downregulations of Mcl-1, Bcl-xl, and Bcl-2 led to the activation of apoptosis of the breast cancer cells. Inhibition of autophagy enhanced the cytotoxic effect of TBMS1 via promoting apoptosis. Our results demonstrate the role and mechanism of TBMS1 in activating autophagy, suggesting that inhibition of cytoprotective autophagy may act as a therapeutic strategy to reinforce the activity of TBMS1 against cancers.

Anticancer strategies based on the metabolic profile of tumor cells: therapeutic targeting of the Warburg effect.

Tumor cells rely mainly on glycolysis for energy production even in the presence of sufficient oxygen, a phenomenon termed the Warburg effect, which is the most outstanding characteristic of energy metabolism in cancer cells. This metabolic adaptation is believed to be critical for tumor cell growth and proliferation, and a number of onco-proteins and tumor suppressors, including the PI3K/Akt/mTOR signaling pathway, Myc, hypoxia-inducible factor and p53, are involved in the regulation of this metabolic adaptation. Moreover, glycolytic cancer cells are often invasive and impervious to therapeutic intervention. Thus, altered energy metabolism is now appreciated as a hallmark of cancer and a promising target for cancer treatment. A better understanding of the biology and the regulatory mechanisms of aerobic glycolysis has the potential to facilitate the development of glycolysis-based therapeutic interventions for cancer. In addition, glycolysis inhibition combined with DNA damaging drugs or chemotherapeutic agents may be effective anticancer strategies through weakening cell damage repair capacity and enhancing drug cytotoxicity.