MIOX inhibits autophagy to regulate the ROS -driven inhibition of STAT3/c-Myc-mediated epithelial-mesenchymal transition in clear cell renal cell carcinoma.

The specific mechanism of clear cell renal cell carcinoma (ccRCC) progression, a pathological type that accounts for the highest proportion of RCC, remains unclear. In this study, bioinformatics analysis of scRNA-seq dataset in ccRCC revealed that MIOX was a gene specifically down-regulated in tumor epithelial cells of ccRCC. Analysis of the TCGA database further validated the association between decreased MIOX mRNA levels and ccRCC malignant phenotype and poor prognosis. Immunohistochemistry indicated the down-regulation of MIOX in ccRCC tissues compared to paired adjacent renal tissues, with further down-regulation of MIOX in the primary tumors of patients with primary metastasis compared to those without metastasis. Also, patients with low expression of MIOX showed shorter metastasis-free survival (MFS) compared to those with high MIOX expression. In vitro results showed that overexpression of MIOX in ccRCC cells inhibited the proliferation, migration and invasion and promoted apoptosis. Mechanistically, up-regulation of MIOX inhibited autophagy to elevate the levels of ROS, and thus suppressed STAT3/c-Myc-mediated epithelial-mesenchymal transition in ccRCC cells. In vivo data further confirmed that increased MIOX expression suppressed the growth and proliferation of RCC cells and reduced the ability of RCC cells to form metastases in the lung. This study demonstrates that MIOX is an important regulatory molecule of ccRCC, which is conducive to understanding the potential molecular mechanism of ccRCC progression.

WDR4 promotes the progression and lymphatic metastasis of bladder cancer via transcriptional down-regulation of ARRB2.

Lymph node (LN) metastasis is one of the key prognostic factors in bladder cancer, but its underlying mechanisms remain unclear. Here, we found that elevated expression of WD repeat domain 4 (WDR4) in bladder cancer correlated with worse prognosis. WDR4 can promote the LN metastasis and proliferation of bladder cancer cells. Mechanistic studies showed that WDR4 can promote the nuclear localization of DEAD-box helicase 20 (DDX20) and act as an adaptor to bind DDX20 and Early growth response 1 (Egr1), thereby inhibiting Egr1-promoted transcriptional expression of arrestin beta 2 (ARRB2) and ultimately contributing to the progression of bladder cancer. Immunohistochemical analysis confirmed that WDR4 expression is also an independent predictor of LN metastasis in bladder cancer. Our results reveal a novel mechanism of LN metastasis and progression in bladder cancer and identify WDR4 as a potential therapeutic target for metastatic bladder cancer.

Stress granules affect the sensitivity of renal cancer cells to sorafenib by sequestering and stabilizing COX­2 mRNA.

Most patients with renal cancer will develop resistance to sorafenib therapy and will therefore exhibit disease progression. Effective therapies for these patients are extremely limited. Cyclooxygenase-2 (COX-2) promotes the malignant transformation of cancer cells and drug resistance. The potential of COX-2 inhibitor (celecoxib) administration in combination with sorafenib for the treatment of renal cancer is unclear. The present study demonstrated that sorafenib rapidly increased the expression of COX-2 in renal cancer cells, as determined using reverse transcription-quantitative PCR and western blotting. The results of the MTT assay and cell apoptosis experiment demonstrated that the cytotoxicity of sorafenib was also affected by COX-2 expression and celecoxib enhanced the cytotoxicity of sorafenib against renal cell carcinoma. Immunofluorescence analysis indicated that sorafenib induced the formation of stress granules (SGs) in renal cancer cells. In addition, COX-2 expression was associated with the formation of SGs, and SGs could capture and stabilize COX-2 mRNAs in renal cancer cells; this was confirmed using RNA fluorescence in situ hybridization and an actinomycin D chase experiment. The protective effect of SGs was further demonstrated in cell experiments and xenograft tumor models. Thus, the present study indicated that the use of celecoxib may significantly enhance the sensitivity of renal cancer cells to sorafenib and improve efficacy. Sorafenib-induced SGs may contribute to critical events that promote COX-2 expression and survival in renal cancer cells. Therefore, the present study may provide novel ideas for the treatment of renal cancer.

CD16+ fibroblasts foster a trastuzumab-refractory microenvironment that is reversed by VAV2 inhibition.

The leukocyte Fcγ receptor (FcγR)-mediated response is important for the efficacy of therapeutic antibodies; however, little is known about the role of FcγRs in other cell types. Here we identify a subset of fibroblasts in human breast cancer that express CD16 (FcγRIII). An abundance of these cells in HER2+ breast cancer patients is associated with poor prognosis and response to trastuzumab. Functionally, upon trastuzumab stimulation, CD16+ fibroblasts reduce drug delivery by enhancing extracellular matrix stiffness. Interaction between trastuzumab and CD16 activates the intracellular SYK-VAV2-RhoA-ROCK-MLC2-MRTF-A pathway, leading to elevated contractile force and matrix production. Targeting of a Rho family guanine nucleotide exchange factor, VAV2, which is indispensable for the function of CD16 in fibroblasts rather than leukocytes, reverses desmoplasia provoked by CD16+ fibroblasts. Collectively, our study reveals a role for the fibroblast FcγR in drug resistance, and suggests that VAV2 is an attractive target to augment the effects of antibody treatments.

Simultaneous treatment with sorafenib and glucose restriction inhibits hepatocellular carcinoma in vitro and in vivo by impairing SIAH1-mediated mitophagy.

Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical significance to explore the synergistic effect of TACE with antiangiogenic inhibitors and the molecular mechanisms involved. This study determined that glucose, but not other analyzed nutrients, offered significant protection against cell death induced by sorafenib, as indicated by glucose deprivation sensitizing cells to sorafenib-induced cell death. Next, this synergistic effect was found to be specific to sorafenib, not to lenvatinib or the chemotherapeutic drugs cisplatin and doxorubicin. Mechanistically, sorafenib-induced mitophagy, as indicated by PINK1 accumulation, increased the phospho-poly-ubiquitination modification, accelerated mitochondrial membrane protein and mitochondrial DNA degradation, and increased the amount of mitochondrion-localized mKeima-Red engulfed by lysosomes. Among several E3 ubiquitin ligases tested, SIAH1 was found to be essential for inducing mitophagy; that is, SIAH1 silencing markedly repressed mitophagy and sensitized cells to sorafenib-induced death. Notably, the combined treatment of glucose restriction and sorafenib abolished ATP generation and mitophagy, which led to a high cell death rate. Oligomycin and antimycin, inhibitors of electron transport chain complexes, mimicked the synergistic effect of sorafenib with glucose restriction to promote cell death mediated via mitophagy inhibition. Finally, inhibition of the glucose transporter by canagliflozin (a clinically available drug used for type-II diabetes) effectively synergized with sorafenib to induce HCC cell death in vitro and to inhibit xenograft tumor growth in vivo. This study demonstrates that simultaneous treatment with sorafenib and glucose restriction is an effective approach to treat HCC, suggesting a promising combination strategy such as transarterial sorafenib-embolization (TASE) for the treatment of unresectable HCC.

Macrophage mitochondrial fission improves cancer cell phagocytosis induced by therapeutic antibodies and is impaired by glutamine competition.

Phagocytosis is required for the optimal efficacy of many approved and promising therapeutic antibodies for various malignancies. However, the factors that determine the response to therapies that rely on phagocytosis remain largely elusive. Here, we demonstrate that mitochondrial fission in macrophages induced by multiple antibodies is essential for phagocytosis of live tumor cells. Tumor cells resistant to phagocytosis inhibit mitochondrial fission of macrophages by overexpressing glutamine-fructose-6-phosphate transaminase 2 (GFPT2), which can be targeted to improve antibody efficacy. Mechanistically, increased cytosolic calcium by mitochondrial fission abrogates the phase transition of the Wiskott-Aldrich syndrome protein (WASP)-Wiskott-Aldrich syndrome interacting protein (WIP) complex and enables protein kinase C-θ (PKC-θ) to phosphorylate WIP during phagocytosis. GFPT2-mediated excessive use of glutamine by tumor cells impairs mitochondrial fission and prevents access of PKC-θ to compartmentalized WIP in macrophages. Our data suggest that mitochondrial dynamics dictate the phase transition of the phagocytic machinery and identify GFPT2 as a potential target to improve antibody therapy.

Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma.

Sorafenib is the first-line medication for advanced hepatocellular carcinoma (HCC), but it can only extend limited survival. It is imperative to find a combination strategy to increase sorafenib efficacy. Artesunate is such a preferred candidate, because artesunate is clinically well-tolerated and more importantly both drugs can induce ferroptosis through different mechanisms. In this study we investigated the combined effect of sorafenib and artesunate in inducing ferroptosis of HCC and elucidated the involved molecular mechanisms. We showed that artesunate greatly enhanced the anticancer effects of low dose of sorafenib against Huh7, SNU-449, and SNU-182 HCC cell lines in vitro and against Huh7 cell xenograft model in Balb/c nude mice. The combination index method confirmed that the combined effect of sorafenib and artesunate was synergistic. Compared with the treatment with artesunate or sorafenib alone, combined treatment induced significantly exacerbated lipid peroxidation and ferroptosis, which was blocked by N-acetyl cysteine and ferroptosis inhibitors liproxstatin-1 and deferoxamine mesylate, but not by inhibitors of other types of cell death (z-VAD, necrostatin-1 and belnacasan). In Huh7 cells, we demonstrated that the combined treatment induced oxidative stress and lysosome-mediated ferritinophagy, two essential aspects of ferroptosis. Sorafenib at low dose mainly caused oxidative stress through mitochondrial impairments and SLC7A11-invovled glutathione depletion. Artesunate-induced lysosome activation synergized with sorafenib-mediated pro-oxidative effects by promoting sequential reactions including lysosomal cathepsin B/L activation, ferritin degradation, lipid peroxidation, and consequent ferroptosis. Taken together, artesunate could be repurposed to sensitize sorafenib in HCC treatment. The combined treatment can be easily translated into clinical applications.

ACSL4 is a predictive biomarker of sorafenib sensitivity in hepatocellular carcinoma.

Sorafenib is the first-line treatment of advanced hepatocellular carcinoma (HCC). However, there is a lack of validated biomarkers to predict sorafenib sensitivity. In this study we investigated the role of ACSL4, a positive-activating enzyme of ferroptosis, in sorafenib-induced cell death and HCC patient outcome. We showed that ACSL4 protein expression was negatively associated with IC50 values of sorafenib in a panel of HCC cell lines (R = -0.952, P < 0.001). Knockdown of ACSL4 expression by specific siRNA/sgRNA significantly attenuated sorafenib-induced lipid peroxidation and ferroptosis in Huh7 cells, and also rescued sorafenib-induced inhibition of xenograft tumor growth in vivo. We selected 29 HCC patients with surgery as primary treatment and sorafenib as postoperative adjunct therapy from a hospital-based cohort. A high proportion (66.7%) of HCC patients who had complete or partial responses to sorafenib treatment (according to the revised RECIST guideline) had higher ACSL4 expression in the pretreated HCC tissues, compared with those who had stable or progressed tumor growth (23.5%, P = 0.029). Since ACSL4 expression was independent of sorafenib treatment, it could serve as a useful predictive biomarker. Taken together, this study demonstrates that ACSL4 is essential for sorafenib-induced ferroptosis and useful for predicting sorafenib sensitivity in HCC. This study may have important translational impacts in precise treatment of HCC.

Quercetin Induces Apoptosis via Downregulation of Vascular Endothelial Growth Factor/Akt Signaling Pathway in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive haematological malignancy characterized by highly proliferative accumulation of immature and dysfunctional myeloid cells. Quercetin (Qu), one kind of flavonoid, exhibits anti-cancer property in multiple types of solid tumor, but its effect on acute myeloid leukemia is less studied, and the underlying mechanisms still largely unknown. This study aimed to explore the specific target and potential mechanism of quercetin-induced cell death in AML. First, we found that quercetin induces cell death in the form of apoptosis, which was caspase dependent. Second, we found that quercetin-induced apoptosis depends on the decrease of mitochondria membrane potential (MMP) and Bcl-2 proteins. With quantitative chemical proteomics, we observed the downregulation of VEGFR2 and PI3K/Akt signaling in quercetin-treated cells. Consistently, cell studies also identified that VEGFR2 and PI3K/Akt signaling pathways are involved in the action of quercetin on mitochondria and Bcl-2 proteins. The decrease of MMP and cell death could be rescued when PI3K/Akt signaling is activated, suggesting that VEGFR2 and PI3K/Akt exert as upstream regulators for quercetin effect on apoptosis induction in AML cells. In conclusion, our findings from this study provide convincing evidence that quercetin induces cell death via downregulation of VEGF/Akt signaling pathways and mitochondria-mediated apoptosis in AML cells.

Corrigendum: Quercetin Induces Apoptosis Via Downregulation of VEGF/Akt Signaling Pathway in Acute Myeloid Leukemia Cells.

[This corrects the article DOI: 10.3389/fphar.2020.534171.].

Histone deacetylases up-regulate C/EBPα expression through reduction of miR-124-3p and miR-25 in hepatocellular carcinoma.

BACKGROUND: CCAAT enhancer binding protein α (C/EBPα), as an important transcription factor involved in cell proliferation, differentiation and metabolism, was up-regulated in primary hepatocellular carcinoma (HCC) and predicted poorer prognosis. In this study, we explored how histone deacetylases (HDACs) up-regulated C/EBPα in HCC. METHODS: The protein expressions of HDAC1, HDAC2 were associated with C/EBPα by immunohistochemistry staining in a HCC tissue microarray. HCC cells were then treated with HDAC inhibitors or siRNAs to determine the roles of miR-124-3p and miR-25 in the regulation of C/EBPα mRNA expression. RESULTS: Both HDAC1 and HDAC2 proteins were significantly associated with C/EBPα. Inhibition of HDAC by either pharmacological inhibitors or siRNAs decreased C/EBPα mRNA expression in dose-dependent manners in HCC cells. HDAC inhibitors reduced C/EBPα mRNA stability as shown by pmiRGLO luciferase reporter assays. HDAC inhibition consistently induced miR-124-3p and miR-25 expression. Conversely, blockage of miR-124-3p and/or miR-25 by treatment with specific synthetic inhibitors abolished C/EBPα reduction. More importantly, C/EBPα mRNA stability could be rescued by site-directed mutations of miR-124-3p or miR-25 recognition sites in the C/EBPα 3'UTR sequence. In summary, HDAC may up-regulate C/EBPα expression through miR-124-3p and miR-25 in HCC.