Chemotherapy-mediated p53-dependent DNA damage response in clear cell renal cell carcinoma: role of the mTORC1/2 and hypoxia-inducible factor pathways

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel–Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.     

Macrophage migration inhibitory factor activates hypoxia-inducible factor in a p53-dependent manner

Background

Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner.

Methods/Principal Findings

The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly.

Conclusion/Significance

MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF''s potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.     

Dapagliflozin,SGLT2 Inhibitor,Attenuates Renal Ischemia-Reperfusion Injury

Dapagliflozin, a new type of drug used to treat diabetes mellitus (DM), is a sodium/glucose cotransporter 2 (SGLT2) inhibitor. Although some studies showed that SGLT2 inhibition attenuated reactive oxygen generation in diabetic kidney the role of SGLT2 inhibition is unknown. We evaluated whether SLT2 inhibition has renoprotective effects in ischemia-reperfusion (IR) models. We evaluated whether dapagliflozin reduces renal damage in IR mice model. In addition, hypoxic HK2 cells were treated with or without SGLT2 inhibitor to investigate cell survival, the apoptosis signal pathway, and the induction of hypoxia-inducible factor 1 (HIF1) and associated proteins. Dapagliflozin improved renal function. Dapagliflozin reduced renal expression of Bax, renal tubule injury and TUNEL-positive cells and increased renal expression of HIF1 in IR-injured mice. HIF1 inhibition by albendazole negated the renoprotective effects of dapagliflozin treatment in IR-injured mice. In vitro, dapagliflozin increased the expression of HIF1, AMP-activated protein kinase (AMPK), and ERK and increased cell survival of hypoxic HK2 cells in a dose-dependent manner. In conclusion, dapagliflozin attenuates renal IR injury. HIF1 induction by dapagliflozin may play a role in renoprotection against renal IR injury.     

Expression of Ror2 Mediates Invasive Phenotypes in Renal Cell Carcinoma

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.     

Hippo Signaling Mediates Proliferation,Invasiveness, and Metastatic Potential of Clear Cell Renal Cell Carcinoma

Recent work has identified dysfunctional Hippo signaling to be involved in maintenance and progression of various human cancers, although data on clear cell renal cell carcinoma (ccRCC) have been limited. Here, we provide evidence implicating aberrant Hippo signaling in ccRCC proliferation, invasiveness, and metastatic potential. Nuclear overexpression of the Hippo target Yes-associated protein (YAP) was found in a subset of patients with ccRCC. Immunostaining was particularly prominent at the tumor margins and highlighted neoplastic cells invading the tumor-adjacent stroma. Short hairpin RNA-mediated knockdown of YAP significantly inhibited proliferation, migration, and anchorage-independent growth of ccRCC cells in soft agar and led to significantly reduced murine xenograft growth. Microarray analysis of YAP knockdown versus mock-transduced ccRCC cells revealed down-regulation of endothelin 1, endothelin 2, cysteine-rich, angiogenic inducer, 61 (CYR61), and c-Myc in ccRCC cells as well as up-regulation of the cell adhesion molecule cadherin 6. Signaling pathway impact analysis revealed activation of the p53 signaling and cell cycle pathways as well as inhibition of mitogen-activated protein kinase signaling on YAP down-regulation. Our data suggest CYR61 and c-Myc as well as signaling through the endothelin axis as bona fide downstream effectors of YAP and establish aberrant Hippo signaling as a potential therapeutic target in ccRCC.     

PBRM1 deficiency oncogenic addiction is associated with activated AKT–mTOR signalling and aerobic glycolysis in clear cell renal cell carcinoma cells

The PBRM1 (PB1) gene which encodes the specific subunit BAF180 of the PBAF SWI/SNF complex, is highly mutated (~ 40%) in clear cell renal cell carcinoma (ccRCC). However, its functions and impact on cell signalling are still not fully understood. Aerobic glycolysis, also known as the ‘Warburg Effect’, is a hallmark of cancer, whether PB1 is involved in this metabolic shift in clear cell renal cell carcinoma remains unclear. Here, with established stable knockdown PB1 cell lines, we performed functional assays to access the effects on 786‐O and SN12C cells. Based on the RNA‐seq data, we selected some genes encoding key glycolytic enzymes, including PFKP, ENO1, PKM and LDHA, and examined the expression levels. The AKT–mTOR signalling pathway activity and expression of HIF1α were also analysed. Our data demonstrate that PB1 deficiency promotes the proliferation, migration, Xenograft growth of 786‐O and SN12C cells. Notably, knockdown of PB1 activates AKT–mTOR signalling and increases the expression of key glycolytic enzymes at both mRNA and protein levels. Furthermore, we provide evidence that deficient PB1 and hypoxic conditions exert a synergistic effect on HIF 1α expression and lactate production. Thus, our study provides novel insights into the roles of tumour suppressor PB1 and suggests that the AKT–mTOR signalling pathway, as well as glycolysis, is a potential drug target for ccRCC patients with deficient PB1.     

Heterogeneous Effects of Direct Hypoxia Pathway Activation in Kidney Cancer

General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumor suppressor. However HIF-1α and HIF-2α have contrasting effects on experimental tumor progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1α and HIF-2α and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of canonical and non-canonical HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1α-specific binding, and genes associated with favorable prognosis and between HIF-2α-specific binding and adverse prognosis. However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-α isoform contributes a highly complex mix of pro- and anti-tumorigenic effects.     

Macrophage migration inhibitory factor increases cell motility and up-regulates αvβ3 integrin in human chondrosarcoma cells

The macrophage migration-inhibitory factor (MIF) is a pro-inflammatory cytokine first known for its effect on macrophage migration and activation. Recent studies have shown that MIP plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of MIF on human chondrosarcoma cells are largely unknown. In the present study, MIF was found to increase the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by MIF treatment, and the MIF-induced expression of integrin and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. In addition, migration-prone sublines demonstrated that increased cell migration ability was correlated with increased expression of MIF and αvβ3 integrin. Taken together, our results indicate that MIF enhanced the migration of the chondrosarcoma cells by increasing αvβ3 integrin expression through the PI3K/Akt/NF-κB signal transduction pathway.     

The Oncogene HER2/neu (ERBB2) Requires the Hypoxia-inducible Factor HIF-1 for Mammary Tumor Growth and Anoikis Resistance

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.     

Tetralone derivatives are MIF tautomerase inhibitors and attenuate macrophage activation and amplify the hypothermic response in endotoxemic mice

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine playing crucial role in immunity. MIF exerts a unique tautomerase enzymatic activity that has relevance concerning its multiple functions and its small molecule inhibitors have been proven to block its pro-inflammatory effects. Here we demonstrate that some of the E-2-arylmethylene-1-tetralones and their heteroanalogues efficiently bind to MIF’s active site and inhibit MIF tautomeric (enolase, ketolase activity) functions. A small set of the synthesised derivatives, namely compounds (4), (23), (24), (26) and (32), reduced inflammatory macrophage activation. Two of the selected compounds (24) and (26), however, markedly inhibited ROS and nitrite production, NF-κB activation, TNF-α, IL-6 and CCL-2 cytokine expression. Pre-treatment of mice with compound (24) exaggerated the hypothermic response to high dose of bacterial endotoxin. Our experiments suggest that tetralones and their derivatives inhibit MIF’s tautomeric functions and regulate macrophage activation and thermal changes in severe forms of systemic inflammation.     

MiR-506 Is Down-Regulated in Clear Cell Renal Cell Carcinoma and Inhibits Cell Growth and Metastasis via Targeting FLOT1

Background

Some microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC).

Methods

miR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines.

Results

miR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506.

Conclusions

miR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.