Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

miR-125a regulates HAS1 and inhibits the proliferation,invasion and metastasis by targeting STAT3 in non–small cell lung cancer cells

MicroRNA-125a (miR-125a) is related to the occurrence, development, and prognosis of various cancers according to relevant reports. However, its function role and mechanism in non–small cell lung cancer (NSCLC) is yet to be explored. Herein, we investigated the role and preliminary mechanism of miR-125a in NSCLC. First, miR-125a was noticeably downregulated in NSCLC tissues in contrast to adjacent normal tissues through the real-time quantitative polymerase chain reaction (RT-qPCR) assay. The inverted result was observed on the STAT3 and HAS1 expressions. Moreover, miR-125a was expressed at highest level in A549 among four human NSCLC cell lines. Second, functional studies indicated miR-125a restrained proliferation, invasion, migration, metastasis, and advocated apoptosis of NSCLC cells, but had no obvious effect on cell cycle. Next, results indicated that a target of miR-125a was STAT3 on the basis of prediction and confirmation by the dual-luciferase reporter assay. RT-qPCR and Western blot assays displayed that miR-125a overexpression conspicuously constrained STAT3 expression at messenger RNA and protein levels. Finally, the binding between HAS1 promoter region and STAT3 was predicted by PROMO database analysis and verified by chromatin immunoprecipitation assay, suggesting that STAT3 was bound with the HAS1 promoter regions. STAT3 overexpression exerted positive effects on HAS1 expression at protein and mRNA levels. Additionally, HAS1-related functional studies illustrated HAS1 pronouncedly suppressed the proliferative, invasive, and migratory potential of NSCLC cells in vitro. Collectively, our findings demonstrated that miR-125a prohibited the proliferation, invasion, and migration of NSCLC cells by HAS1 expression reduction as a result of inhibiting STAT3 expression in NSCLC. This study indicated that miR-125a might be of potential or value for NSCLC treatment.     

Long noncoding RNA FEZF1-AS1 predicts poor prognosis and modulates pancreatic cancer cell proliferation and invasion through miR-142/HIF-1α and miR-133a/EGFR upon hypoxia/normoxia

Nowadays, pancreatic cancer (PC) remains the most lethal tumor, partially due to the invasive and treatment-resistant phenotype induced by the extent of hypoxic stress within the tumor tissue. According to previous studies, miR-142/HIF-1α and miR-133a/EGFR could modulate PC cell proliferation under hypoxic and normoxic conditions, respectively. In the present study, FEZF1-AS1, a recently described oncogenic long noncoding RNA, was predicted to target both miR-142 and miR-133a; thus, we hypothesized that FEZF1-AS1 might affect PC cell proliferation through these two axes under hypoxic or normoxic conditions. In PC cell lines, FEZF1-AS1 acted as an oncogene via promoting PC cell proliferation and invasion through miR-142/HIF-1α axis under hypoxic condition; however, FEZF1-AS1 failed to affect the protein levels of HIF-1α and VEGF under the normoxic condition, suggesting the existence of another signaling pathway under normoxic condition. As predicted by an online tool, FEZF1-AS1 could target miR-133a to inhibit its expression; under the normoxic condition, FEZF1-AS1 exerted its effect on PC cell lines through miR-133a/EGFR axis. Taken together, FEZF1-AS1 might be a promising target in controlling the aberrant proliferation and invasion of PC cell lines.     

HIF-1α signaling by airway epithelial cell K-α1-tubulin: role in fibrosis and chronic rejection of human lung allografts

Long term survival of the human lung allografts are hindered by chronic rejection, manifested clinically as bronchiolitis obliterans syndrome (BOS). We previously demonstrated significant correlation between the development of antibodies (Abs) to K-α1-tubulin (Kα1T) and BOS. In this study, we investigated the molecular basis for fibrinogenesis mediated by ligation of Kα1T expressed on airway epithelial cells by its specific Abs. Using RT-PCR we demonstrate that normal human bronchial epithelial (NHBE) cells upon ligation of Kα1T with specific Abs caused upregulation of pro-fibrotic growth factors. Western blot analysis of NHBE incubated with Kα1T Abs increased hypoxia inducible factor (HIF-1α). Kα1T Ab-mediated growth factor expression is dependent on HIF-1α as inhibition of HIF-1α returned fibrotic growth factor expression to basal levels. In conclusion, we propose that HIF-1α -mediated upregulation of fibrogenic growth factors induced by ligation of Kα1T Abs is critical for development of fibrosis leading to chronic rejection of lung allograft.     

A drug targeting only p110α can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110β/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.     

miR-155 inhibits the formation of hypertrophic scar fibroblasts by targeting HIF-1α via PI3K/AKT pathway

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by the excessive proliferation of fibroblasts and often considered as a kind of benign skin tumor. microRNA-155 (miR-155) is usually served as a promising marker in antitumor therapy. In view of the similarities of hypertrophic scar and tumor, it is predicted that miR-155 may be a novel therapeutic target in clinical trials. Here we found the expression levels of miR-155 was gradually down regulated and HIF-1α was upregulated in HS tissue and HS derived fibroblasts (HFs). And cell proliferation was inhibited when miR-155 was overexpressed or HIF-1α was silenced. Moreover, overexpression of miR-155 in HFs could reduce the expression of collagens in vitro and inhibit the collagen fibers arrangement in vivo, whereas miR-155 knockdown gave opposite results. Furthermore, we found that miR-155 directly targeted the HIF-1α, which could also independently inhibit the expression of collagens in vitro and obviously improved the appearance and architecture of the rabbit ear scar in vivo when it was silencing. Finally, we found that PI3K/AKT pathway was enrolled in these processes. Together, our results indicated that miR-155 was a critical regulator in the formation and development of hypertrophic scar and might be a potential molecular target for hypertrophic scar therapy.     

Britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by targeting PD-L1 via abrogation of the crosstalk between Myc and HIF-1α in cancer

BackgroundProgrammed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which causes tumor cells to escape T cell killing, and promotes tumor cell survival, cell proliferation, migration, invasion, and angiogenesis. Britannin is a natural product with anticancer pharmacological effects.PurposeIn this work, we studied the anticancer potential of britannin and explored whether britannin mediated its effect by inhibiting the expression of PD-L1 in tumor cells.MethodsIn vitro, the mechanisms underlying the inhibition of PD-L1 expression by britannin were investigated by MTT assay, homology modeling and molecular docking, RT-PCR, western blotting, co-immunoprecipitation, and immunofluorescence. The changes in tumor killing activity, cell proliferation, cell cycle, migration, invasion, and angiogenesis were analyzed by T cell killing assays, EdU labeling, colony formation, flow cytometry, wound healing, matrigel transwell invasion, and tube formation, respectively. In vivo, the antitumor activity of britannin was evaluated in the HCT116 cell xenograft model.ResultsBritannin reduced the expression of PD-L1 in tumor cells by inhibiting the synthesis of the PD-L1 protein but did not affect the degradation of the PD-L1 protein. Britannin also inhibited HIF-1α expression through the mTOR/P70S6K/4EBP1 pathway and Myc activation through the Ras/RAF/MEK/ERK pathway. Mechanistically, britannin inhibited the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. In addition, britannin could enhance the activity of cytotoxic T lymphocytes and inhibit tumor cell proliferation and angiogenesis by inhibiting PD-L1. Finally, in vivo observations were confirmed by demonstrating the antitumor activity of britannin in a murine xenograft model.ConclusionBritannin inhibits the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. Moreover, britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by inhibiting PD-L1 in cancer. The current work highlights the anti-tumor effect of britannin, providing insights into the development of cancer therapeutics via PD-L1 inhibition.     

miR-193b-5p regulates chondrocytes metabolism by directly targeting histone deacetylase 7 in interleukin-1β-induced osteoarthritis

There is increasing evidence regarding the pivotal roles of microRNAs (miRNAs) and histone deacetylases (HDACs) in the development of osteoarthritis (OA). This study aimed to determine whether miR-193b-5p regulates HDAC7 expression directly to affect cartilage degeneration. Expression levels of miR-193b-5p, HDAC7, matrix metalloproteinase 3 (MMP3), and MMP13 were determined in normal and OA cartilage and primary human chondrocytes (PHCs) stimulated with interleukin-1β (IL-1β). PHCs were transfected with a miR-193b-5p mimic or inhibitor to verify whether miR-193b-5p influences the expression of HDAC7 and MMPs. A luciferase reporter assay was performed to demonstrate the binding between miR-193b-5p and the 3′-untranslated region (UTR) of HDAC7. Expression of miR-193b-5p was reduced in IL-1β-stimulated PHCs and in OA cartilage compared to that in normal cartilage. Luciferase reporter assay exhibited the repressed activity of the reporter construct containing the 3′UTR of HDAC7. Both miR-193b-5p overexpression and HDAC7 inhibition decreased the expression of MMP3 and MMP13, whereas the inhibition of miR-193b-5p enhanced HDAC7, MMP3, and MMP13 expression. miR-193b-5p downregulates HDAC7 directly and, as a result, inhibits MMP3 and MMP13 expression, which suggests that miR-193b-5p has a protective role in OA.