Chun-Min Ma

Important Role of SUMOylation of Spliceosome Factors in Prostate Cancer Cells

Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells.

Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, β-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.

Autophagy inhibition enhances isobavachalcone-induced cell death in multiple myeloma cells.

Despite recent advancements in therapeutic drugs, multiple myeloma remains an incurable disease. Therefore, a more effective treatment is urgently required. In this study, we show that isobavachalcone (IBC), a natural chalcone compound, induces apoptosis- and autophagy-related cell death in myeloma cells. The inhibition of autophagy by knocking down beclin-1 or by using autophagy inhibitors, such as 3-methyladenine, bafilomycin A and chloroquine significantly enhanced IBC-induced cell death, as demonstrated by the increased number of Annexin V-positive cells. Moreover, we demonstrate that the collapse of the mitochondrial membrane potential contributes to chloroquine and IBC-induced cell death, which is accompanied by the activation of caspase-9, and -3, the cleavage of poly (ADP-ribose) polymerase (PARP) and the proteolytic activation of protein kinase Cδ (PKCδ). Furthermore, the inhibition of the activation of PKCδ by rottlerin, an inhibitor of PKCδ, not only suppressed the activation of PKCδ, but also the apoptosis induced by the co-treatment of chloroquine and IBC, indicating the involvement of PKCδ in chloroquine plus IBC-induced cell death. Finally, the combination of chloroquine and IBC had little effect on the viability of normal peripheral blood mononuclear cells. As both chloroquine and IBC have been shown to be relatively specific for cancer cells, the combination of these two agents at non-toxic or sub-toxic concentrations represents an attractive novel regimen for myeloma treatment and warrants further investigation in preclinical and clinical studies.

Ikaros inhibits proliferation and, through upregulation of Slug, increases metastatic ability of ovarian serous adenocarcinoma cells

The transcription factor Ikaros was originally found to function as a key regulator of lymphocyte differentiation. In this study, we provide the first evidence that Ikaros is expressed at higher levels in ovarian cancer tissues compared with normal ovarian tissues and is significantly associated with high FIGO stage and low differentiation state in ovarian serous adenocarcinoma. To this end, we transfected IK1 (full length of Ikaros) into the SKOV3 ovarian cancer cell line and examined cell biological behaviors including proliferation, migration and invasion. We found that overexpression of IK1 inhibited cell proliferation by inducing G1 arrest, accompanied by the upregulation of P27 and P21 and downregulation of cyclin D1 and D2. On the other hand, IK1 increased the migration and invasion of ovarian cancer cells, as assessed by scratch-wound assay, transwell migration assay, and invasion assay. Overexpression of IK1 significantly increased Slug but not Snail1 expression at both mRNA and protein levels. It also downregulated and upregulated E-cadherin and MMP-2, two target genes of Slug involved in migration, respectively. Furthermore, knocking down Slug abrogated IK1-mediated increase in migration and invasion. These data suggest that Slug plays an important role in IK1-induced migration and invasion. In conclusion, we show for the first time that IK1 plays a dual role in the proliferation, migration and invasion of ovarian cancer cells, providing new insights into their metastasis.

Targeting catalase but not peroxiredoxins enhances arsenic trioxide-induced apoptosis in K562 cells.

Despite considerable efficacy of arsenic trioxide (As2O3) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As2O3 treatment. However, the underlying mechanism is not well understood. Here we show that relative As2O3-resistant K562 cells have significantly lower ROS levels than As2O3-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As2O3. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As2O3. On the contrary, knockdown of catalase markedly enhanced As2O3-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As2O3 in CML treatment.

Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells

Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

Identification of H7 as a novel peroxiredoxin I inhibitor to induce differentiation of leukemia cells.

Identifying novel targets to enhance leukemia-cell differentiation is an urgent requirment. We have recently proposed that inhibiting the antioxidant enzyme peroxiredoxin I (Prdx I) may induce leukemia-cell differentiation. However, this concept remains to be confirmed. In this work, we identified H7 as a novel Prdx I inhibitor through virtual screening, in vitro activity assay, and surface plasmon resonance assay. Cellular thermal shift assay showed that H7 directly bound to Prdx I but not to Prdxs II–V in cells. H7 treatment also increased reactive oxygen species (ROS) level and cell differentiation in leukemia cells, as reflected by the upregulation of the cell surface differentiation marker CD11b/CD14 and the morphological maturation of cells. The differentiation-induction effect of H7 was further observed in some non-acute promyelocytic leukemia (APL) and primary leukemia cells apart from APL NB4 cells. Moreover, the ROS scavenger N-acetyl cysteine significantly reversed the H7-induced cell differentiation. We demonstrated as well that H7-induced cell differentiation was associated with the activation of the ROS-Erk1/2-C/EBPβ axis. Finally, we showed H7 treatment induced cell differentiation in an APL mouse model. All of these data confirmed that Prdx I was novel target for inducing leukemia-cell differentiation and that H7 was a novel lead compound for optimizing Prdx I inhibition.

YL064 directly inhibits STAT3 activity to induce apoptosis of multiple myeloma cells

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) plays a critical role in the proliferation and survival of multiple myeloma. And inactivation of STAT3 is considered a promising strategy for the treatment of multiple myeloma. Here we show that the sinomenine derivative YL064 could selectively reduce the cell viability of multiple myeloma cell lines and primary multiple myeloma cells. Moreover, YL064 also induces cell death of myeloma cells in the presence of stromal cells. Western blot analysis showed that YL064 inhibited the constitutive activation and IL-6-induced activation of STAT3, reflected by the decreased phosphorylation of STAT3 on Tyr705. Consistent with this, YL064 inhibited the nuclear translocation of STAT3 and the expression of STAT3 target genes, such as cyclin D1 and Mcl-1. Using biotin- and FITC-labeled YL064, we found that YL064 could pull-down STAT3 from myeloma cells and colocalized with STAT3, suggesting that YL064 directly targets STAT3. Cellular thermal shift assay further demonstrated the engagement of YL064 to STAT3 in cells. Molecular docking studies indicated that YL064 may interact with STAT3 in its SH2 domain, thereby inhibiting the dimerization of STAT3. Finally, YL064 inhibited the growth of human myeloma xenograft in vivo. Taken together, this study demonstrated that YL064 may be a promising candidate compound for the treatment of multiple myeloma by directly targeting STAT3.

Identification of 11(13)-dehydroivaxillin as a potent therapeutic agent against non-Hodgkin's lymphoma

Despite great advancements in the treatment of non-Hodgkin lymphoma (NHL), sensitivity of different subtypes to therapy varies. Targeting the aberrant activation NF-κB signaling pathways in lymphoid malignancies is a promising strategy. Here, we report that 11(13)-dehydroivaxillin (DHI), a natural compound isolated from the Carpesium genus, induces growth inhibition and apoptosis of NHL cells. Multiple signaling cascades are influenced by DHI in NHL cells. PI3K/AKT and ERK are activated or inhibited in a cell type dependent manner, whereas NF-κB signaling pathway was inhibited in all the NHL cells tested. Applying the cellular thermal shift assay, we further demonstrated that DHI directly interacts with IKKα/IKKβ in NHL cells. Interestingly, DHI treatment also reduced the IKKα/IKKβ protein level in NHL cells. Consistent with this finding, knockdown of IKKα/IKKβ inhibits cell proliferation and enhances DHI-induced proliferation inhibition. Overexpression of p65, p52 or RelB partially reverses DHI-induced cell growth inhibition. Furthermore, DHI treatment significantly inhibits the growth of NHL cell xenografts. In conclusion, we demonstrate that DHI exerts anti-NHL effect in vitro and in vivo, through a cumulative effect on NF-κB and other pathways. DHI may serve as a promising lead compound for the therapy of NHL.

Sinomenine derivative YL064: a novel STAT3 inhibitor with promising anti-myeloma activity

Multiple myeloma (MM) is the second most common form of blood cancer. The introduction of proteasome inhibitors (e.g., velcade), immunomodulators (e.g., lenalidomide), and other novel agents have greatly improved the prognosis of patients with MM. However, recurrence or drug resistance occurs frequently. In particular, the bone marrow microenvironment confers protection effect to MM cells by direct cell contact or releasing cytokines such as interleukin-6 (IL-6). Finding novel treatment is urgently needed. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates the expression of many genes, such as Bcl-xL, Mcl-1, and Cyclin D1. STAT3 is involved in variety of biological processes, such as cell proliferation, differentiation, survival, inflammatory response, immunity, and angiogenesis. In MM cells, STAT3 is aberrant activated by endogenous (e.g., IKK) or exogenous signals (e.g., stromal cells or IL-6) (Fig. 1). Targeting STAT3 is considered as a promising strategy against MM. Sinomenine is a natural compound isolated from Sinomeniumacutum and has been used clinically for the treatment of rheumatoid arthritis and other inflammatory diseases. However, in order to achieve efficacy, sinomenine must be used at high concentrations, which causes some side effects and limits its application. In a recent article published in Cell Death Discovery, we synthesized a series of sinomenine derivatives and identified YL064 as the most effective one. YL064 is at least 10-fold more potent than sinomenine in exerting cytotoxic effect against MM cells, but is not cytotoxic to normal blood cells even at higher concentrations. Importantly, YL064 can induce MM cell death in the presence of stromal cells or in the addition of exogenous IL-6. Furthermore, in vivo experiments shown that YL064 is well tolerated in mice and it could significantly inhibit tumor growth. These data suggest that YL064 is a promising lead compound for the treatment of MM. Due to the pivotal role of STAT3 in the pathogenesis of MM, we examined the possible effect of YL064 on STAT3 activation. Interestingly, YL064 can inhibit the phosphorylation of Tyr705 but not Ser727, indicating the suppression of STAT3 activity. In support of this, YL064 inhibited the nuclear translocation of STAT3 and the transcription of STAT3 target genes, including cyclin D1, Mcl-1. Intriguingly, both the endogenous activation and exogenous factorsinduced activation of STAT3 could be blocked by YL064. Based on these data, we hypothesized that YL064 may directly interacts with STAT3. For this purpose, we synthesized Biotin-labelled or FITC-labelled YL064. The results showed that Biotin-YL064 could pull down STAT3 from cell lysate, which could be competed away by unlabeled YL064, indicating the specific interaction between STAT3 and YL064. And FITC-YL064 co-localized with STAT3 in MM cells. Moreover, using the cellular thermal shift assay, we further demonstrated the engagement of YL064 with STAT3 in cells. Taken together, our data strongly suggest that