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DJ-1 is a multifunctional protein associated with cancers and autosomal early-onset Parkinson disease. Besides the well-documented antioxidative stress activity, recent studies show that DJ-1 has deglycation enzymatic activity and anti-ferroptosis effect. It has been shown that DJ-1 forms the homodimerization, which dictates its antioxidative stress activity. In this study, we investigated the relationship between the dimeric structure of DJ-1 and its newly reported activities. In HEK293T cells with Flag-tagged and Myc-tagged DJ-1 overexpression, we performed deletion mutations and point mutations, narrowed down the most critical motif at the C terminus. We found that the deletion mutation of the last three amino acids at the C terminus of DJ-1 (DJ-1 ΔC3) disrupted its homodimerization with the hydrophobic L187 residue being of great importance for DJ-1 homodimerization. In addition, the ability in methylglyoxal (MGO) detoxification and deglycation was almost abolished in the mutation of DJ-1 ΔC3 and point mutant L187E compared with wild-type DJ-1 (DJ-1 WT). We also showed the suppression of erastin-triggered ferroptosis in DJ-1(−/−) mouse embryonic fibroblast cells was abolished by ΔC3 and L187E, but partially diminished by V51C. Thus, our results demonstrate that the C terminus of DJ-1 is crucial for its homodimerization, deglycation activity, and suppression of ferroptosis.

Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding motif (TAZ), play pivotal roles in promoting the progression of hepatocellular carcinoma. However, the regulatory mechanism underpinning aberrant activation of YAP/TAZ in hepatocellular carcinoma remains unclear. In this study, we globally profiled the contribution of deubiquitinating enzymes (DUB) to both transcriptional activity and protein abundance of YAP/TAZ in hepatocellular carcinoma models and identified ubiquitin-specific peptidase 10 (USP10) as a potent YAP/TAZ-activating DUB. Mechanistically, USP10 directly interacted with and stabilized YAP/TAZ by reverting their proteolytic ubiquitination. Depletion of USP10 enhanced polyubiquitination of YAP/TAZ, promoted their proteasomal degradation, and ultimately arrested the proliferation of hepatocellular carcinoma in vitro and in vivo. Expression levels of USP10 positively correlated with the abundance of YAP/TAZ in hepatocellular carcinoma patient samples as well as in N-nitrosodiethylamine (DEN)-induced liver cancer mice models. Collectively, this study establishes the causal link between USP10 and hyperactivated YAP/TAZ in hepatocellular carcinoma cells and provides a rationale for potential therapeutic interventions in the treatment of patients with hepatocellular carcinoma harboring a high level of YAP/TAZ. Significance: These findings identify USP10 as a DUB of YAP/TAZ and its role in hepatocellular carcinoma progression, which may serve as a potential therapeutic target for hepatocellular carcinoma treatment.

Various lipids and lipid metabolites are bound to and modify the proteins in eukaryotic cells, which are known as ‘protein lipidation’. There are four major types of the protein lipidation, i.e. myristoylation, palmitoylation, prenylation, and glycosylphosphatidylinositol anchor. N-myristoylation refers to the attachment of 14-carbon fatty acid myristates to the N-terminal glycine of proteins by N-myristoyltransferases (NMT) and affects their physiology such as plasma targeting, subcellular tracking and localization, thereby influencing the function of proteins. With more novel pathogenic N-myristoylated proteins are identified, the N-myristoylation will attract great attentions in various human diseases including infectious diseases, parasitic diseases, and cancers. In this review, we summarize the current understanding of N-myristoylation in physiological processes and discuss the hitherto implication of crosstalk between N-myristoylation and other protein modification. Furthermore, we mention several well-studied NMT inhibitors mainly in infectious diseases and cancers and generalize the relation of NMT and cancer progression by browsing the clinic database. This review also aims to highlight the further investigation into the dynamic crosstalk of N-myristoylation in physiological processes as well as the potential application of protein N-myristoylation in translational medicine.

Hepatocellular carcinoma (HCC) has emerged as one of the most prevalent life‐threatening cancers, and the high mortality rate is largely due to the metastasis. The sustained activation of Smad4 and transforming growth factor‐β (TGF‐β) is closely associated with advanced HCC metastasis. However, the regulatory mechanism underlying the aberrant activation of Smad4 and TGF‐β pathway remains elusive. In this study, using a functional screen of USPs siRNA library, we identified ubiquitin‐specific proteases USP10 as a deubiquitinating enzyme (DUB) that sustains the protein level of Smad4 and activates TGF‐β signaling. Further analysis showed that USP10 directly interacts with Smad4 and stabilizes it through the cleavage of its proteolytic ubiquitination, thus promoting HCC metastasis. The suppression of USP10 by either shRNAs or catalytic inhibitor Spautin‐1 significantly inhibited the migration of HCC cells, whereas the reconstitution of Smad4 was able to efficiently rescue this defect. Overall, our study not only uncovers the regulatory effect of USP10 on the protein abundance of Smad4, but also indicates that USP10 could be regarded as a potential intervention target for the metastatic HCC in Smad4‐positive patients., Ubiquitin‐specific protease USP10 was shown to activate TGF‐β signaling and promote the metastasis of advanced HCC by deubiquitinating and stabilizing Smad4. Depletion of USP10 or inhibition of the USP10 catalytic activity by use of the small‐molecule inhibitor Spautin‐1 significantly repressed HCC metastasis, whereas reconstitution with Smad4 could efficiently rescue the metastatic phenotype.

Aldo-keto reductase family 1 member C1 (AKR1C1) promotes malignancy of Non-Small Cell Lung Cancer (NSCLC) by activating Signal Transducer and Activator of Transcription 3 (STAT3) pathway. However, how the pro-metastatic functions of AKR1C1 are switched on/off remains unknown. Methods: Immunoprecipitation and LC-MS/MS analyses were performed to identify the acetylation on AKR1C1 protein, and the functional analyses (in vitro and in vivo) were performed to depict the contribution of acetylation to the pro-metastatic effects of AKR1C1. Results: Here we report that acetylated AKR1C1 on two lysine residues K185 & K201 is critical to its pro-metastatic role. The acetylation modification has no impact on the canonical enzymatic activity of AKR1C1, while it is required for the interaction between AKR1C1 to STAT3, which triggers the downstream transduction events, ultimately mobilizing cells. Importantly, the deacetylase Sirtuin 2 (SIRT2) is capable of deacetylating AKR1C1, inhibiting the transactivation of STAT3 target genes, thus suppressing the migration of cells. Conclusion: Acetylation on Lysines 185 and 201 of AKR1C1 dictates its pro-metastatic potential both in vitro and in vivo, and the reverting of acetylation by Sirtuin 2 provides potential therapeutic targets for treatment against metastatic NSCLC patients with high AKR1C1 expression.

Stress granules (SGs) are primarily composed of mRNAs that stall at translation initiation and usually appear in the cytoplasm under unusual physiological or pathological conditions such as hypoxia, oxidative stress, and viral infection. Recent studies have indicated that several components of SGs participate in tumourigenesis and cancer metastasis through tumour‐associated signalling pathways as well as other mechanisms. Furthermore, some chemotherapy drugs have been reported to induce SGs. Thus, the roles of SGs in cancer treatment have attracted considerable interest. Importantly, disturbing the recruitment of SGs components or microtubule polymerization, as well as other strategies that can abolish SGs formation, is reported to inhibit tumour progression, suggesting that targeting SGs could be a promising strategy for cancer treatment. In this review, we summarize the relationship between SGs and cancer, as well as recent advances in targeting SGs, in the interest of providing new opportunities for cancer treatment.

Insufficient pancreatic β-cell mass or insulin-producing β-cells are implicated in all forms of diabetes mellitus. However, the molecular mechanisms underlying β-cell destruction are complex and not fully defined. Here we observed that activation of STAT3 is intensely and specifically inhibited in β-cells under hyperglycemic conditions. By knocking out STAT3 specifically in mouse β-cells, we found that the loss of STAT3 sensitized mice to three low doses of STZ stimulation resulting in hyperglycemia. Mechanistically, accumulating PTEN, induced by STAT3 deficiency, directly represses phosphorylation of AKT, which negatively modulates transcription factor activation, dysregulates β-cell function, positively promotes apoptotic signaling, and finally induces β-cell apoptosis. Notably, the defective secretion of insulin and β-cells apoptosis was completely rescued by PTEN ablation in STAT3-null islets or PTEN inhibitor bpv(phen) treatment. Thus our data suggest that STAT3 is a vital modulator of β-cell survival and function, highlighting a critical role for STAT3 in the negative regulation of PTEN-AKT signaling pathway associated with β-cell dysfunction and apoptosis.

Background: Cellular senescence is generally understood as a permanent cell cycle arrest stemming from different causes. The mechanism of cellular senescence-induced cell cycle arrest is complex, involving interactions between telomere shortening, inflammations and cellular stresses. In recent years, a growing number of studies have revealed that cellular senescence could mediate the cancer progression of neighboring cells, but this idea is controversial and contradictory evidence argues that cellular senescence also contributes to tumor suppression. Objective: Given that the complicated role of senescence in various physiological and pathological scenarios, we try to clarify the precise contribution role of cellular senescence to tumor progression. Methods: Search for the information in a large array of relevant articles to support our opinion. Results: We discuss the relatively widespread occurrence of cellular senescence in cancer treatment and identify the positive and negative side of senescence contributed to tumor progression. Conclusion: We argue that the availability of pro-senescence therapy could represent as a promising regimen for managing cancer disease, particularly with regard to the poor clinical outcome obtained with other anticancer therapies.

The PARK7 gene (encode DJ-1 protein) was first discovered as an oncogene and later found to be a causative gene for autosomal recessive early onset Parkinson’s disease. DJ-1 has been proposed as a potential therapeutic anticancer target due to its pivotal role in tumorigenesis and cancer progression. Based on the homodimer structure of DJ-1, a series of bis-isatin derivatives with different length linkers were designed, synthesized, and evaluated as dimeric inhibitors targeting DJ-1 homodimer. Among them, DM10 with alkylene chain of C10 displayed the most potent inhibitory activity against DJ-1 deglycase. We further demonstrated that DM10 bound covalently to the homodimer of DJ-1. In human cancer cell lines H1299, MDA-MB-231, BEL7402, and 786-O, DM10 (2.5–20 μM) inhibited the cell growth in a concentration-dependent manner showing better anticancer effects compared with the positive control drug STK793590. In nude mice bearing H1299 cell xenograft, intratumor injection of DM10 (15 mg/kg) produced significantly potent tumor growth inhibition when compared with that caused by STK793590 (30 mg/kg). Moreover, we found that DM10 could significantly enhance N-(4-hydroxyphenyl)retinamide-based apoptosis and erastin-based ferroptosis in H1299 cells. In conclusion, DM10 is identified as a potent inhibitor targeting DJ-1 homodimer with the potential as sensitizing agent for other anticancer drugs, which might provide synergistical therapeutic option for cancer treatment.

Ferroptosis is a newly characterized form of regulated cell death mediated by iron-dependent accumulation of lipid reactive oxygen species and holds great potential for cancer therapy. However, the molecular mechanisms underlying ferroptosis remain largely elusive. In this study, we define an integrative role of DJ-1 in ferroptosis. Inhibition of DJ-1 potently enhances the sensitivity of tumor cells to ferroptosis inducers both in vitro and in vivo. Metabolic analysis and metabolite rescue assay reveal that DJ-1 depletion inhibits the transsulfuration pathway by disrupting the formation of the S-adenosyl homocysteine hydrolase tetramer and impairing its activity. Consequently, more ferroptosis is induced when homocysteine generation is decreased, which might be the only source of glutathione biosynthesis when cystine uptake is blocked. Thus, our findings show that DJ-1 determines the response of cancer cells to ferroptosis, and highlight a candidate therapeutic target to potentially improve the effect of ferroptosis-based antitumor therapy., DJ-1 protects cells from oxidative stress-induced cell death; however its role in ferroptosis is unclear. Here, the authors show that DJ-1 suppresses ferroptosis through the transsulfuration pathway to compensate for the depletion of intracellular cysteine when cystine import is inhibited.

Given that Yes-associated protein (YAP) signaling acts as a critical survival input for hypoxic cancer cells in hepatocellular carcinoma (HCC), disruption of YAP function and the maintenance of hypoxia is an attractive way to treat HCC. Utilizing a cell-based YAP-TEAD luciferase reporter assay and functional analyses, we identified CT-707, a China-FDA approved multi-kinase inhibitor under clinical trial with remarkable inhibitory activity against YAP function. CT-707 exhibited prominent cytotoxicity under hypoxia on HCC cells, which was attributable to the inhibition of YAP signaling. CT-707 arrested tumor growth in HepG2, Bel-7402, and HCC patient-derived xenografts. Mechanistically, the inhibitory activity of CT-707 on YAP signaling was due to the interruption of hypoxia-activated IGF1R. Overall, these findings not only identify CT-707 as a promising hypoxia-targeting agent against HCC, but they also unveil IGF1R as a new modulator specifically regulating hypoxia-activated YAP signaling. Significance: CT-707 may represent a novel clinical approach for patients with HCC suffering poor drug response due to intratumor hypoxia. Cancer Res; 78(14); 3995–4006. ©2018 AACR.

A distinct hallmark of acute myeloid leukemia (AML) is the arrest of leukemic myeloblasts at an immature stage of development. Therapies that overcome differentiation arrest have emerged as a powerful strategy for treating AML, but targeting leukemia differentiation remains challenging, mainly because of an incomplete mechanistic understanding of the process. Here, we unveil a new role for cyclin-dependent kinase 2 (CDK2) in blocking myeloid differentiation in AML. We show that among several interphase CDK, only CDK2 undergoes ubiquitin-dependent proteasome degradation, which is accompanied by AML cell differentiation. By using the yeast 2-hybrid system and functional analyses, KLHL6 was identified as a specific E3 ubiquitin ligase regulating the degradation of CDK2. Importantly, inhibiting CDK2, but not other cyclin-dependent kinases CDK1/4/6, effectively induced granulocytic differentiation in AML cell lines and 5 major subtypes of primary patient-derived AML samples. Mechanistically, CDK2 depletion led to the reactivation of differentiation pathway translation, and the differentiation blockade function of CDK2 may be achieved directly by maintaining the activity of PRDX2. Finally, CDK2 depletion arrested tumor growth of AML cells in nude mice and extended survival in both AML cell line and PDX-AML cells derived xenograft mouse models. Thus, our work not only provides experimental evidence for validating CDK2 as a potential therapeutic target for differentiation, but also uncovers the biological function of the CDK2-PRDX2 axis in blocking AML differentiation.

Osteosarcoma, a highly malignant tumor, is characterized by widespread and recurrent chromosomal and genetic abnormalities. In recent years, a number of elaborated sequencing analyses have made it possible to cluster the osteosarcoma based on the identification of candidate driver genes and develop targeted therapy. Here, we reviewed recent next-generation genome sequencing studies and advances in targeted therapies for osteosarcoma based on molecular classification. First, we stratified osteosarcomas into ten molecular subtypes based on genetic changes. And we analyzed potential targeted therapies for osteosarcoma based on the identified molecular subtypes. Finally, the development of targeted therapies for osteosarcoma investigated in clinical trials were further summarized and discussed. Therefore, we indicated the importance of molecular classification on the targeted therapy for osteosarcoma. And the stratification of patients based on the genetic characteristics of osteosarcoma will help to obtain a better therapeutic response to targeted therapies, bringing us closer to the era of personalized medicine.

M2-polarized tumor-associated macrophages (TAM) play a critical role in cancer invasion and metastasis. Here, we report that M2 macrophages enhanced metastasis of K7M2 WT osteosarcoma cells to the lungs in mice, thus establishing M2 TAMs as a therapeutic target for blocking osteosarcoma metastasis. We found that all-trans retinoic acid (ATRA) inhibited osteosarcoma metastasis via inhibiting the M2 polarization of TAMs. ATRA suppressed IL13- or IL4-induced M2-type macrophages, and then inhibited migration of osteosarcoma cells as promoted by M2-type macrophages in vitro. ATRA reduced the number of pulmonary metastatic nodes of osteosarcoma and decreased expression of M2-type macrophages in metastatic nodes both in intravenous injection and orthotopic transplantation models. ATRA's effect was independent of conventional STAT3/6 or C/EBPβ signaling, which regulate M2-like polarization of macrophages. Quantitative genomic and functional analyses revealed that MMP12, a macrophage-secreted elastase, was elevated in IL13-skewed TAM polarization, whereas ATRA treatment downregulated IL13-induced secretion of MMP12. This downregulation correlates with the antimetastasis effect of ATRA. Our results show the role of TAM polarization in osteosarcoma metastasis, identify a therapeutic opportunity for antimetastasis treatment, and indicate ATRA treatment as an approach for preventing osteosarcoma metastasis via M2-type polarization intervention. Cancer Immunol Res; 5(7); 547–59. ©2017 AACR.

Clinical studies have shown that fenretinide (4-HPR) is an attractive chemopreventive agent for cancer treatment. However, to date, few studies have demonstrated the mechanism of the preventive effect of 4-HPR. In our current study, we revealed that 4-HPR could significantly suppress IL-4/IL-13 induced M2-like polarization of macrophages, which was demonstrated by the reduced expression of M2 surface markers, the down-regulation of M2 marker genes, and the inhibition of M2-like macrophages promoted angiogenesis. Mechanistically, our study suggested that the inhibition of the phosphorylation of STAT6, rather than the generation of oxidative stress, is involved in the 4-HPR-driven inhibition of M2 polarization. More intriguingly, by utilizing adenomatous polyposis coli (APCmin/+) transgenic mice, we demonstrated that the tumorigenesis was dramatically decreased by 4-HPR treatment accompanied with fewer M2-like macrophages in the tumor tissues, thereby profoundly blocking tumor angiogenesis. These findings, for the first time, reveal the involvement of M2 polarization inhibition in 4-HPR-mediated chemoprevention, which provides a new point of insight and indicates the potential mechanism underlying the chemopreventive effect of 4-HPR.

Kelch-like gene family members (KLHLs) encode proteins with a bric-a-brac, tramtrack, broad complex (BTB)/poxvirus and zinc finger (POZ) domain, a BACK domain, and six Kelch motifs, which frequently interact with Cullin3 to form E3 ligase complexes that mediate the ubiquitination of substrate proteins. In recent years, studies have revealed that mutations and abnormal expression of KLHLs are closely related to the pathogenesis of various human diseases. Increasing evidence has shown that Kelch-like protein family members (KLHLs) exert important biological functions through the ubiquitination of specific substrates. This review provides an overview of the identified substrates of different KLHLs, summarizes the current knowledge of KLHLs and discusses the biological functions of KLHLs in different diseases.

Differentiation therapy involves the use of agents that can induce differentiation in cancer cells, with the irreversible loss of tumour phenotype. The application of differentiation therapy in osteosarcoma has made progress because of a better understanding of the potential links between differentiation defects and tumorigenesis. Here, we review recent studies on differentiation therapy for osteosarcoma, describing a variety of differentiation inducers. By highlighting these examples of drug-induced osteosarcoma cell differentiation, we can acquire unique insights into not only osteosarcoma treatment, but also novel approaches to transform differentiating drugs into more effective therapies for other solid tumours.

Emerging evidence indicates that M2-polarized tumor-associated macrophages (TAMs) directly participate in tumor initiation, progression and metastasis. However, to date, few studies have investigated novel strategies for inhibiting TAMs in order to overcome osteosarcoma. In this study, we reported that M2 macrophages were enriched in osteosarcoma tissues from patients, and M2-polarized TAMs enhanced cancer initiation and stemness of osteosarcoma cells, thereby establishing M2-polarized TAMs as a therapeutic target for blocking osteosarcoma formation. We also found that all-trans retinoic acid (ATRA) weakened TAM-induced osteosarcoma tumor formation by inhibiting M2 polarization of TAMs in vivo, and inhibited the colony formation, as well as sphere-formation capacity of osteosarcoma cells promoted by M2-type macrophages in vitro. Furthermore, M2-type macrophages enhanced cancer stem cells (CSCs) properties as assessed by increasing the numbers of CD117(+)Stro-1(+) cells accompanied by the upregulation of CSC markers (CD133, CXCR4, Nanog, and Oct4), which could clearly be reduced by ATRA. Taken together, the results of this study demonstrated the role of M2-polarized TAMs in osteosarcoma initiation and stemness by activating CSCs, and indicated that ATRA treatment is a promising approach for treating osteosarcoma by preventing M2 polarization of TAMs.

A characteristic feature of leukemia cells is a blockade of differentiation in cellular maturation. All-trans-retinoic acid (ATRA) has been successfully applied for the treatment of M3-type AML (APL, 10 %), but it fails to demonstrate a significant efficacy on the remaining patients with non-APL AML (90 %). Therefore, the research for strategies to extend the efficacy of ATRA-based therapy to non-APL AML is a key avenue of investigation. Here, we evaluate the synergetic effect of CDK2 inhibition and ATRA in AML both in vitro and in vivo. We have determined that both the CDK2 depletion and pharmacological inhibitor of CDK2 significantly sensitize three subtypes of AML cells (including two non-APL cells) to ATRA-induced cell differentiation. RNA-sequence results indicate that transcription activation of differentiation and maturation pathways plays an important role in this synergetic effect. Furthermore, the down-regulation of CDK2 sensitized AML cells to ATRA-induced engraftment prevention of leukemia cells in NOD-SCID mice and promotes the primary AML blasts differentiation when combined with ATRA. Thus, our work not only provides relevant experimental evidence for further validating CDK2 as a target for differentiation therapy, but also uncovers the future clinical application of CDK2 inhibitors in ATRA-based differentiation therapeutics for AML.

Despite more effective chemotherapy combined with limb-salvage surgery for the osteosarcoma treatment, survival rates for osteosarcoma patients have stagnated over the past three decades due to the poor prognosis. Osteosarcoma cancer stem cells (OSCs) are responsible for the growth and metastasis of osteosarcoma. The existence of OSCs offers a theoretical explanation for therapeutic failures including tumor recurrence, metastasis, and drug resistance. Understanding the pathways that regulate properties of OSCs may shed light on mechanisms that lead to osteosarcoma and suggest better modes of treatment. In this study, we showed that the expression level of Kruppel-like factor 4 (KLF4) is highly associated with human osteosarcoma cancer stemness. KLF4-overexpressed osteosarcoma cells displayed characteristics of OSCs: increased sphere-forming potential, enhanced levels of stemness-associated genes, great chemoresistance to adriamycin and CDDP, as well as more metastasis potential. Inversely, KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo, supporting an oncogenic role for KLF4 in osteosarcoma pathogenesis. Furthermore, KLF4 was shown to activate the p38 MAPK signaling pathway to promote cancer stemness. Altogether, our studies uncover an essential role for KLF4 in regulation of OSCs and identify KLF4–p38 MAPK axis as a potential therapeutic target for osteosarcoma treatment.

M2 polarization of macrophages is essential for their function in immunologic tolerance, which might promote tumorigenesis. However, the molecular mechanism behind the polarization process is not fully understood. Given that several lines of evidence have suggested that long noncoding RNAs (lncRNAs) could be involved in regulating immune cell differentiation and function, the current study aimed to identify the lncRNAs that specifically modulate M2 macrophage polarization. By utilizing a series of cell-based M2 macrophage polarization models, a total of 25 lncRNAs with altered expression were documented based on lncRNA microarray-based profiling assays. Among them, lncRNA-MM2P was the only lncRNA upregulated during M2 polarization but downregulated in M1 macrophages. Knockdown of lncRNA-MM2P blocked cytokine-driven M2 polarization of macrophages and weakened the angiogenesis-promoting feature of M2 macrophages by reducing phosphorylation on STAT6. Moreover, manipulating lncRNA-MM2P in macrophages impaired macrophage-mediated promotion of tumorigenesis, tumor growth in vivo, and tumor angiogenesis. Collectively, our study identifies lncRNA-MM2P as a modulator required for macrophage M2 polarization and uncovers its role in macrophage-promoted tumorigenesis.

DJ-1 is a gene involved in various cellular processes, including transcriptional regulation, oxidative stress response, fertilization, mitochondrial regulation, inflammatory and fibrogenic niche formation, and glycation damage prevention. Although a disease-associated genetic study within the past decade has demonstrated that the mutation of DJ-1 is associated with autosomal early-onset Parkinson's disease, increasing evidence suggests that DJ-1 also plays a critical role in tumor development and progression. In this review, we provide an overview of current knowledge concerning the role and the mechanism of DJ-1 in cancer and also discuss the possibility of DJ-1 as a therapeutic target against cancer.

Osteosarcoma is the most common bone cancer, and chemotherapy is currently indispensable for its treatment. Adriamycin has been claimed to be the most effective agent for osteosarcoma, however, the outcome of adriamycin chemotherapy remains unsatisfactory. Here, we reported a potent combination therapy that bortezomib, a proteasome inhibitor, enhances adriamycin-induced apoptosis to eliminate osteosarcoma cells and we revealed that the activation of p-eIF2α/ATF4/CHOP axis is the underlying associated mechanisms. First, we observed that bortezomib enhances adriamycin-mediated inhibition of cell proliferation and enhances the apoptosis in osteosarcoma cell lines. Moreover, this drug combination produced more potent tumor-growth inhibitory effects in human osteosarcoma cell line KHOS/NP xenografts. Our study showed that reactive oxygen species (ROS) plays an important role in apoptosis induced by adriamycin plus bortezomib, whereas ROS scavenger NAC could almost completely block the apoptosis induced by the combination treatment. Meanwhile, p-eIF2α is remarkably elevated in the combination group. As a result, ATF4 exhibits strong activation which consequently induces the activation of CHOP and leads to the cell death. Finally, 13 primary osteosarcoma cells demonstrated potent response to the combination treatment. In a human osteosarcoma patient-derived xenograft (PDX) model, our finding suggests that when combined with bortezomib, a relatively low dose of adriamycin produced more potent tumor-growth inhibitory effects without increased toxicity. Thus, our findings not only provide a promising combination strategy to overcome osteosarcoma but also shed new light on the strategy of combining increased ROS and inhibited proteasome to open up new opportunities for the clinical development of chemotherapy regimens.

Small ubiquitin-related modifier-1 (SUMO-1) modification has been implicated in many important cellular processes, including cell cycle progression, apoptosis, cellular proliferation, and development, but its role in all-trans-retinoic acid (ATRA)-induced differentiation processes of cancer cells remains unclear. Here, we report for the first time that ATRA-induced differentiation of leukemia and osteosarcoma is accompanied by a decrease in the level of SUMO-1 protein. Our results also demonstrated that depletion or inhibition of SUMO-1 blocks ATRA-induced differentiation, suggesting that SUMO-1 is critical for the differentiation effect of ATRA. Further studies indicated that SUMO-1-promoted ATRA-induced differentiation might be associated with the stabilization of retinoic acid receptor α (RARα), protecting it from degradation. Moreover, our results suggested that Lys399 is a major site for SUMO-1 conjugation of RARα. We also found that RARα enhanced the transcription of its target genes, which might also contribute to the enhanced differentiating effects of ATRA; however, mutation of Lys399 of RARα inhibits the extents of both SUMO-1 modification and ATRA-induced differentiation. Together, these results indicate that SUMO-1 modification of RARα is a potent mechanism for balancing proliferation and differentiation by controlling the stability of RARα in cancer cells. SUMO-1 modification may thus serve an important role in controlling ATRA-induced cell differentiation in cancers.

Hypoxia is a common phenomenon occurring in the majority of human tumors and has been proved to play an important role in tumor progression. However, it remains unclear that whether the action of hypoxia on macrophages is a main driving force of hypoxia-mediated aggressive tumor behaviors. In the present study, we observe that high density of M2 macrophages is associated with metastasis in adenocarcinoma Non-Small Cell Lung Cancer (NSCLC) patients. By applying the in vivo hypoxia model, the results suggest that intermittent hypoxia significantly promotes the metastasis of Lewis lung carcinoma (LLC), accompanied with more CD209+ macrophages infiltrated in primary tumor tissue. More intriguingly, by skewing macrophages polarization away from the M1- to a tumor-promoting M2-like phenotype, hypoxia and IL-6 cooperate to enhance the LLC metastasis both in vitro and in vivo. In addition, we also demonstrate that skewing of macrophage M2 polarization by hypoxia relies substantially on activation of ERK signaling. Collectively, these observations unveil a novel tumor hypoxia concept involving the macrophage phenotype shift and provide direct evidence for lung cancer intervention through modulating the phenotype of macrophages.

Neohesperidin (NHP), derived from citrus fruits, has attracted considerable interest due to its preventative and therapeutic effects on numerous diseases. However, little progress has been made in determining the exact function of NHP on tumorigenesis. In the current study, we found that NHP inhibited colorectal tumorigenesis in the APC min/+ transgenic mouse model, as well as induced apoptosis and blocked angiogenesis in vivo. Our in-cell study suggested that this tumorigenic preventative effect of NHP is not due to the direct impact on tumor cells. Intriguingly, by utilizing 16 s rRNA gene-based microbiota sequencing, the relative abundance of Bacteroidetes was decreased, while Firmicutes and Proteobacteria were increased in the presence of NHP. Additionally, the fecal microbiota transplantation experiment further revealed that feeding with fecal of NHP-treated mice induced considerable inhibition of tumorigenesis, which indicates that the alteration of gut microbiota is responsible for NHP-mediated prevention of colorectal tumorigenesis. Thus, our study not only suggests the efficacy of NHP as a potent natural product for preventing colorectal cancer but also proposes a compelling model to connect the gut microbiota to the preventative effect of NHP on tumorigenesis.

Nickel, a metal commonly found in battery plants and welding factories, has potential cardiotoxicity, while all-trans retinoid acid (atRA) can promote cardiovascular repair and myocardial recovery. The purpose of this study was to investigate whether atRA could prevent cardiotoxicity induced by nickel both in vitro and in vivo. In the study, a rat myocardial cell line (H9c2) exposed to different concentrations of nickel chloride (NiCl(2)) displayed apoptotic features accompanied by reactive oxygen species generation. In addition, NiCl(2) also caused obvious apoptosis and systolic dysfunction in primary myocardial cells. Treatment with atRA efficiently attenuated the cytotoxicities triggered by NiCl(2) as it significantly mitigated ROS generation and decreased MAP kinases activity in NiCl(2)-treated cardiomyocytes. Additionally, NiCl(2) exposure caused obvious arrhythmia in Sprague-Dawley rats with the maximum tolerance dose of NiCl(2) between 2 and 3mg/kg. A combinational intragastric administration of 40mg/kg atRA can partially reverse NiCl(2)-induced arrhythmia in rats. Our results suggested that atRA might have therapeutic potential in alleviating the adverse effects of nickel on the cardiovascular system.

Sorafenib is a multikinase inhibitor used as a first-line treatment for advanced hepatocellular carcinoma (HCC), but it has shown modest to low response rates. The characteristic tumour hypoxia of advanced HCC maybe a major factor underlying hypoxia-mediated treatment failure. Thus, it is urgent to elucidate the mechanisms of hypoxia-mediated sorafenib resistance in HCC. In this study, we found that hypoxia induced the nuclear translocation of Yes associate-Protein (YAP) and the subsequent transactivation of target genes that promote cell survival and escape apoptosis, thereby leading to sorafenib resistance. Statins, the inhibitors of hydroxymethylglutaryl-CoA reductase, could ameliorate hypoxia-induced nuclear translocation of YAP and suppress mRNA levels of YAP target genes both in vivo and in vitro. Combined treatment of statins with sorafenib greatly rescued the loss of anti-proliferative effects of sorafenib under hypoxia and improved the inhibitory effects on HepG2 xenograft tumour growth, accompanied by enhanced apoptosis as evidenced by the increased sub-G1 population and PARP cleavage. The expression levels of YAP and its target genes were highly correlated with poor prognosis and predicted a high risk of HCC patients. These findings collectively suggest that statins utilization maybe a promising new strategy to counteract hypoxia-mediated resistance to sorafenib in HCC patients.

Acriflavine (ACF), a known antibacterial drug, has recently been recognized as a suitable candidate for cancer chemotherapy. However, the molecular target of ACF is not fully understood, which limits its application in cancer therapy. In this study, we established a structure-specific probe-based pull-down approach to comprehensively profile the potential target of ACF, and we identified DNA dependent protein kinase catalytic subunit (DNA-PKcs) as the direct target of ACF. Since DNA-PKcs facilitates the repair process following DNA double-strand breaks, we further developed a drug combination strategy that combined ACF with the bifunctional alkylating agent melphalan, which exerted a p53-dependent synergistic efficacy against human cancer cells both in vitro and in vivo. With these findings, our study demonstrated that structure-specific probe-based pull-down approaches can be used to identify new functional target of drug, and provided novel opportunities for the development of ACF-based antitumor chemotherapies.

Hepatocellular carcinoma is among the leading causes of cancer-related deaths worldwide, and the development of new treatment regimens is urgently needed to improve therapeutic approach. In our study, we found that the combination of a Met inhibitor, cabozantinib, and a novel FAK inhibitor, CT-707, exerted synergistic antitumor effects against hepatocellular carcinoma in vitro and in vivo. Interestingly, further studies showed that therapeutic concentrations of cabozantinib increased the phosphorylation of FAK, which might attenuate the antitumor activity of cabozantinib. The simultaneous exposure to CT-707 effectively inhibited the activation of FAK that was induced by cabozantinib, which contributes to the synergistic effect of the combination. Furthermore, cabozantinib increased the mRNA and protein levels of integrin α5, which is a canonical upstream of FAK, and the introduction of cilengitide to block integrin function could abrogate FAK activation by cabozantinib, indicating that cabozantinib upregulated the phosphorylation of FAK in an integrin-dependent manner. Similar synergy was also observed on PHA-665752, another selective MET inhibitor, indicating that this observation might be a common characteristic of MET-targeting strategies. Our findings not only favor the development of the novel FAK inhibitor CT-707 as a therapeutic agent against hepatocellular carcinoma but also provide a new strategy of combining MET and FAK inhibitors to potentiate the anticancer activities of these two types of agents for treating hepatocellular carcinoma patients. Mol Cancer Ther; 15(12); 2916–25. ©2016 AACR.

The biological relationships among self-renewal, tumorigenicity and lineage differentiation of human osteosarcoma-initiating cells (OSIC) remain elusive, making it difficult to identify and distinguish OSIC from osteosarcoma-forming cells (OSFC) for developing OSIC-targeted therapies. Using a new inverse-lineage tracking strategy coupled with serial human-to-mouse xenotransplantation, we identified a subpopulation of osteosarcoma cells with OSIC-like properties and sought to distinguish them from their progeny, OSFC. We found that serial transplantation of cells from different osteosarcoma cell lines and primary osteosarcoma tissues progressively increased the CD49f(+) subpopulation composing the bulk of the osteosarcoma mass. These CD49f(+) cells displayed characteristics of OSFC: limited in vivo tumorigenicity, weak lineage differentiation, more differentiated osteogenic feature and greater chemo-sensitivity. By contrast, their parental CD49f(-)CD133(+) cells had an inhibited osteogenic fate, together with OSIC-like properties of self-renewal, strong tumorigenicity and differentiation to CD49f(+) progeny. Hence, the CD49f(-)CD133(+) phenotype appears to identify OSIC-like cells that possess strong tumorigenicity correlated with an impaired osteogenic fate and the ability to initiate tumor growth through the generation of CD49f(+) progeny. These findings advance our understanding of OSIC-like properties and, for the first time, provide a much-needed distinction between OSIC and OSFC in this cancer.

// Ning Zhang 1 , Tao Xie 1 , Miao Xian 2 , Yi-Jie Wang 2 , Heng-Yuan Li 1 , Mei-Dan Ying 2, * , Zhao-Ming Ye 1, * 1 Department of Orthopaedics, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, P.R. China 2 Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, P.R. China * These authors have contributed equally to this work Correspondence to: Mei-Dan Ying, email: remember319@hotmail.com Zhao-Ming Ye, email: zrorthopaedics@gmail.com Keywords: osteosarcoma, metastasis, SIRT1, invasion, biomarker Received: December 29, 2015 Accepted: September 25, 2016 Published: October 26, 2016 ABSTRACT Pulmonary metastasis is the leading cause of mortality in patients with osteosarcoma; however, the underlying mechanism remains unclear. The NAD + -dependent deacetylase, sirtuin 1 (SIRT1), has been reported to play a key role in carcinogenesis through deacetylation of important regulatory proteins. Here, we report that SIRT1 promotes osteosarcoma metastasis by regulating the expression of metastatic-associated genes. The SIRT1 protein was significantly upregulated in most primary osteosarcoma tumours, compared with normal tissues, and the SIRT1 expression level may be coupled with metastatic risk in patients with osteosarcoma. Moreover, the results of cell migration and wound-healing assays further suggested that higher expression of SIRT1 promoted invasive activity of osteosarcoma cells. Importantly, downregulating SIRT1 with shRNA inhibited the migration ability of osteosarcoma cells in vitro and suppressed tumour lung metastasis in mice. Finally, a gene expression analysis showed that knockdown of SIRT1 profoundly activated translation of its downstream pathway, particularly at migration and invasion. In summary, high levels of SIRT1 may be a biomarker for a high metastatic rate in osteosarcoma patients; inhibiting SIRT1 could be a potent therapeutic intervention for these patients.

Intratumoral hypoxia occurs in many solid tumors, where it is associated with the development of metastatic character. However, the connections between these phenomena are not fully understood. In this study, we define an integrative role for the E3 ubiquitin ligase subunit WSB1. In primary osteosarcomas, increased levels of WSB1 correlated with pulmonary metastatic potential. RNAi-mediated attenuation of WSB1 or disruption of its E3 ligase activity potently suppressed tumor metastasis. Quantitative proteomic and functional analyses revealed that WSB1 ubiquitylates the Rho-binding protein RhoGDI2 and promotes its proteasomal degradation, thereby activating Rac1 to stimulate tumor cell motility and invasion. Our findings show how WSB1 regulates key steps of the metastatic cascade in hypoxia-driven osteosarcoma, and they highlight a candidate therapeutic target to potentially improve the survival of patients with metastatic disease. Cancer Res; 75(22); 4839–51. ©2015 AACR.

DJ-1 is a cancer- and Parkinson's disease-associated protein that participates in different intracellular signaling pathways to protect cells from toxic stresses. DJ-1 expression, oxidation, localization, and phosphorylation are often altered in human tumors, and DJ-1 has been implicated in various aspects of transformation, including uncontrolled proliferation, invasion, metastasis, and resistance to chemotherapy and apoptosis. Despite the strong relationship between DJ-1 and cancer, which made it a particularly attractive therapeutic target for cancer treatment, the detailed mechanisms of how this oncogene coordinates altered signaling with cell survival remains elusive. In this commentary, we discuss the role of DJ-1 in transformation, highlight some of the significant aspects of and prospects for therapeutically targeting the DJ-1 signaling in cancer, and describe what the future may hold.

Although SAHA is approved for the treatment of cutaneous T-cell lymphoma by the U.S. Food and Drug Administration, clinical trials using SAHA as a monotherapy or in combination with other chemotherapeutic agents in solid tumors have not met with success, and the mechanisms of tolerance remain unknown. In this study, using the prostate cancer cell line PC3 and the non-small lung cancer cell line A549, which have limited sensitivity to SAHA, we found that SAHA triggered MET and AKT phosphorylation at clinical concentrations. siRNA silencing of MET enhanced SAHA induced apoptosis in PC3 and A549 cells. However, MET protein expression and HGF secretion were not affected by SAHA, suggesting that the SAHA-induced MET activation was not due to MET over-expression or HGF paracrine secretion. However, mRNA and protein expression of the laminin receptor integrin α5β1 was up-regulated by SAHA prior to MET activation. Silencing of integrin α5β1 abolished SAHA-triggered MET phosphorylation, suggesting the involvement of integrin α5β1 in MET activation. Further, the combination of SAHA and XL184 resulted in a synergistic induction of cancer cell apoptosis and a synergistic inhibition of tumor growth. These data indicate that SAHA triggered MET activation in an HGF independent manner. This effect is partially involved in the resistance to SAHA in solid cancers, warranting further clinical investigation into combining SAHA with MET inhibitors in solid cancer treatment.

Dihydroartemisinin (DHA), one of the main metabolites of artemisinin and its derivatives, presents anti-cancer potential in vitro and in vivo. To explore the mechanisms of resistance toward DHA, a DHA-resistant cell line, HeLa/DHA, was established with a resistance factor of 7.26 in vitro. Upon DHA treatment, apoptotic cells were significantly elicited in parental HeLa cells but minimally induced in HeLa/DHA cells. HeLa/DHA cells also displayed much less sensitivity to DHA-induced tumor suppression in cancer xenograft models than HeLa cells. Intriguingly, DHA-resistant cells did not display a multidrug-resistant phenotype. Based on a proteomic study employing LC-ESI-MS/MS together with pathway analysis, DJ-1 (PARK7) was found to be highly expressed in HeLa/DHA cells. Western blot and immunofluorescence assays confirmed the higher expression of DJ-1 in HeLa/DHA cells than in parental cells in both cell line and xenograft models. DJ-1 is translocated to the mitochondria of HeLa/DHA cells and oxidized, providing DJ-1 with stronger cytoprotection activity. Further study revealed that DJ-1 knockdown in HeLa/DHA cells abolished the observed resistance, whereas overexpression of DJ-1 endowed the parental HeLa cells with resistance toward DHA. Reactive oxygen species (ROS) were also significantly induced by either DHA or hydrogen peroxide in HeLa cells but not in resistant HeLa/DHA cells. When the cells were pretreated with N-acetyl-l-cysteine, the effect of DJ-1 knockdown on sensitizing HeLa/DHA cells to DHA was significantly attenuated. In summary, our study suggests that overexpression and mitochondrial translocation of DJ-1 provides HeLa/DHA cells with resistance to DHA-induced ROS and apoptosis.

All-trans-retinoic acid (ATRA) has held great promise for differentiation-based therapy but reportedly downregulates retinoic acid receptor-α (RARα) in a proteasome-dependent manner, which leads to decreased acute myeloid leukemia (AML) cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond acute promyelocytic leukemia (APL) are key investigative avenues. Here, we show that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes, indicative of myeloid differentiation without cell death. In addition, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (P < 0.05). These enhanced differentiation effects were accompanied by RARα stabilization and STAT1 activation. Taken together, our study was the first to evaluate bortezomib and ATRA synergy in AML cell differentiation and to assess new opportunities for bortezomib and ATRA combination as a promising approach for future differentiation therapy. Mol Cancer Ther; 12(2); 195–206. ©2012 AACR.

Although retinoic acid (RA) is a potent agent that coordinates inhibition of proliferation with differentiation of many cell types, RA-mediated signaling pathways in osteosarcoma cell differentiation are uncharacterized. In this study, we show that in human U2OS osteosarcoma cells, decreased phosphorylation of RA receptor alpha (RARalpha) by RA treatment or overexpressing a phosphorylation-defective mutant RARalphaS77A results in the inhibition of proliferation and induction of differentiation, and that U2OS cells transduced with RARalphaS77A suppresses tumor formation in nude mice. Moreover, using different human primary osteosarcoma cells and human mesenchymal stem cells for gene expression analysis, we found that either RA or RARalphaS77A induces many of the same differentiation response pathways and signaling molecules involved in U2OS cell differentiation. In addition, overexpression of the fibroblast growth factor 8f (FGF8f), one of the downstream targets induced by both RA and RARalphaS77A in U2OS cells, inhibits proliferation and induces expression of osteoblastic differentiation regulators. Hence, these data strongly suggest that RA-suppressed phosphorylation of RARalpha induces FGF8f expression to mediate differentiation response pathway in U2OS osteosarcoma cells.

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver. It is unfortunate that HCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of HCCs. We now report that (+/-)-(3aRS,4SR)-2-(2-chloro-4-methylsulfonylphenyl)-4'-chloro-3alpha,4-diethoxy-flavane[4,3-d]-D1,9b-1,2,3-thiadiazoline (MSFTZ), a synthesized flavanone derivative, induced growth arrest and apoptosis of HCCs both in vitro and in vivo. MSFTZ induced a time- and dose-dependent increase in HCC apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase-1 cleavage. Activation of caspase-9 induced by MSFTZ suggested that MSFTZ-induced signaling was mediated through a mitochondrial death pathway. In addition, we observed an elevation of reactive oxygen species (ROS) and a consequent loss of mitochondrial membrane potential, further suggesting that MSFTZ-induced death signaling was mediated through a mitochondrial oxygen stress pathway. These events were associated with a decrease and increase in Bcl-2 and Bax expression, respectively, as well as phosphorylation of mitogen-activated protein kinase (MAPK) and activation of p53-MDM2 pathway. However, the antioxidant N-acetylcysteine opposed MSFTZ-mediated mitochondrial dysfunction, caspase activation, Bcl-2/Bax modulation, and apoptosis, supporting the role of ROS in the apoptotic process. We were surprised that we failed to observe the protective effect of N-acetylcysteine against MSFTZ-induced MAPK activation. Furthermore, MSFTZ had an antitumor effect in vivo by 34.8 to 78.7% reduction of tumor size in SMMC-7721-xenografted nude mice. We conclude that MSFTZ induces HCC cell apoptosis both in vivo and in vitro via caspase- and ROS-dependent mitochondrial pathway. In addition, MSFTZ has potential as a novel therapeutic agent for the treatment of HCC.

Protracted administration of topotecan (TPT), a topoisomerase I inhibitor, exhibited high anticancer efficacy both in animal models and human cancers. This phenomenon is related to the TPT-induced inhibition of angiogenesis in tumor, but the potential mechanism remains largely unknown. In the present study, we reported that TPT (1-10 microM) could inhibit angiogenesis in a dose-dependent manner in Chick embryo chorioallantoic membrane (CAM) assay. TPT showed strong inhibitory activity against proliferation on human EA.hy926 endothelial cells with an IC50 value of 0.13 microM (MTT assay), lower than that of most sensitive cancer cell lines (IC50 range, 0.17 microM to 5.1 microM). TPT could induce EA.hy926 cells undergoing apoptosis, and the percentage of apoptotic cells induced by TPT (0.05 microM-5.0 microM) were 17.9%-52.3%. The similar results were observed with AO/EB staining. Flow cytometry assay also revealed that various concentrations of TPT induced cell cycle disturbance in EA.hy926 cells. Western blotting results showed that TPT caused an obvious increase of p53 expression and a decline of ERK expression in EA.hy926 cells. In addition, the VEGF expression of PC-3 cells is inhibited by TPT in hypoxia. Altogether, inhibiting proliferation of endothelial cells and down-regulating VEGF expression in cancer cells may involve in the antiangiogenesis mechanism of TPT.

Staphylococcus Aureus Filtrate Preparation (SAFP) is a preparation of low-virulence strain of Staphylococcus aureus. In our study, we aimed to determine the anti-tumor activity of SAFP in vivo and explore the potential mechanism.Our study evaluated the anti-tumor activity of SAFP in four cancer cell models in vivo and determined its up-regulatory effects on mice immune system in vivo.It showed that 7.2 ng/10 g SAFP could inhibit the progression of murine hepatoma H22 and sarcoma S180, and the tumor inhibition rates achieved 48.1 and 35.7%, respectively. In human non-small cell lung cancer A549 and hepatoma BEL7402 xenograft athymic mice models, T/C of 7.2 ng/10 g SAFP group reached 41.0% in A549 model and 21.0% in BEL7402 model. Meanwhile, SAFP also significantly increased the proliferation of cultured mice splenocytes to 162.1%, and 7.2 ng/kg SAFP significantly (P0.001) raised the activity of NK cells, elevated the serum level of IL-2, TNF-alpha and IFN-gamma in mice.SAFP exhibited high efficiency in inhibiting the growth of cancer cells in a dose-dependent manner, which possibly attributed to its regulatory effects of immune system.