Search

High invasive capacity and acquired tyrosine kinase inhibitors (TKI) resistance of kidney renal clear cell carcinoma (KIRC) cells remain obstacles to prolonging the survival time of patients with advanced KIRC. In the present study, we reported that sine oculis homeobox 1 (SIX1) was upregulated in sunitinib-resistant KIRC cells and metastatic KIRC tissues. Subsequently, we found that SIX1 mediated metastasis and sunitinib resistance via Focal adhesion (FA) signaling, and knockdown of SIX1 enhanced the antitumor efficiency of sunitinib in KIRC. Mechanistically, Integrin subunit beta 1 (ITGB1), an upstream gene of FA signaling, was a direct transcriptional target of SIX1. In addition, we showed that DExH-box helicase 9 (DHX9) was an important mediator for SIX1-induced ITGB1 transcription, and silencing the subunits of SIX1/DHX9 complex significantly reduced transcription of ITGB1. Downregulation of SIX1 attenuated nuclear translocation of DHX9 and abrogated the binding of DHX9 to ITGB1 promoter. Collectively, our results unveiled a new signal axis SIX1/ITGB1/FAK in KIRC and identified a novel therapeutic strategy for metastatic KIRC patients., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in cancer, however, posttranscriptional regulation such as N 6 -methyladenosine (m 6 A) of IGF1R remains unclear. Here, we reveal a role for a lncRNA Downregulated RNA in Cancer (DRAIC) suppress tumor growth and metastasis in clear cell Renal Carcinoma (ccRCC). Mechanistically, DRAIC physically interacts with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) and enhances its protein stability by blocking E3 ligase F-box protein 11 (FBXO11)-mediated ubiquitination and proteasome-dependent degradation. Subsequently, hnRNPA2B1 destabilizes m 6 A modified-IGF1R, leading to inhibition of ccRCC progression. Moreover, four m 6 A modification sites are identified to be responsible for the mRNA degradation of IGF1R. Collectively, our findings reveal that DRAIC/hnRNPA2B1 axis regulates IGF1R mRNA stability in an m 6 A-dependent manner and highlights an important mechanism of IGF1R fate. These findings shed light on DRAIC/hnRNPA2B1/FBXO11/IGF1R axis as potential therapeutic targets in ccRCC and build a link of molecular fate between m 6 A-modified RNA and ubiquitin-modified protein., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

While Stimulator-of-interferon genes (STING) is an innate immune adapter cruicial for sensing cytosolic DNA and modulating immune microenvironment, its tumor-promoting role in tumor survival and immune evasion remains largely unknown. Here we reported that renal cancer cells are exceptionally dependent on STING for survival and evading immunosurveillance via suppressing ER stress-mediated pyroptosis. We found that STING is significantly amplified and upregulated in clear cell renal cell carcinoma (ccRCC), and its elevated expression is associated with worse clinical outcomes. Mechanically, STING depletion in RCC cells specifically triggers activation of the PERK/eIF2α/ATF4/CHOP pathway and activates cleavage of Caspase-8, thereby inducing GSDMD-mediated pyroptosis, which is independent of the innate immune pathway of STING. Moreover, animal study revealed that STING depletion promoted infiltration of CD4 + and CD8 + T cells, consequently boosting robust antitumor immunity via pyroptosis-induced inflammation. From the perspective of targeted therapy, we found that Compound SP23, a PROTAC STING degrader, demonstrated comparable efficacy to STING depletion both in vitro and in vivo for treatment of ccRCC. These findings collectively unveiled an unforeseen function of STING in regulating GSDMD-dependent pyroptosis, thus regulating immune response in RCC. Consequently, pharmacological degradation of STING by SP23 may become an attractive strategy for treatment of advanced RCC., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Clear cell renal cell carcinoma (ccRCC) presents a unique profile characterized by high levels of angiogenesis and robust vascularization. Understanding the underlying mechanisms driving this heterogeneity is essential for developing effective therapeutic strategies. This study revealed that ubiquitin B (UBB) is downregulated in ccRCC, which adversely affects the survival of ccRCC patients. UBB exerts regulatory control over vascular endothelial growth factor A (VEGFA) by directly interacting with specificity protein 1 (SP1), consequently exerting significant influence on angiogenic processes. Subsequently, we validated that DNA methyltransferase 3 alpha (DNMT3A) is located in the promoter of UBB to epigenetically inhibit UBB transcription. Additionally, we found that an unharmonious UBB/VEGFA ratio mediates pazopanib resistance in ccRCC. These findings underscore the critical involvement of UBB in antiangiogenic therapy and unveil a novel therapeutic strategy for ccRCC., (© 2024. The Author(s).)

Despite the promise of targeted tyrosine kinase inhibitors (TKIs), such as sunitinib, in the extension of survival time in patients with clear cell renal cell carcinoma (ccRCC) progression or metastasis, the patients eventually succumb to inevitable drug resistance. Protein degradation executed by the ubiquitin-dependent proteasome system played an important role in determining the sensitivity of ccRCC to sunitinib. Here, we applied the bioinformatic analysis to identify that E3 ligase RBCK1 was elevated in the sunitinib-resistant renal cancer cell lines or patient specimens. The subsequent in vitro or in vivo studies demonstrated that RBCK1 contributed to decreasing the sensitivity of ccRCC to sunitinib. Then, we showed that inhibition of RBCK1 inactivated the AKT and MAPK signaling pathways, which might be one of the main reasons why RBCK1 induces sunitinib resistance in ccRCC cells. Mechanistically, our results indicated that RBCK1 promotes the degradation of ANKRD35 and that ANKRD35 destabilizes MITD1 by binding with SUMO2 in ccRCC cells. In addition, we showed that the RBCK1-ANKRD35-MITD1-ANXA1 axis regulates the phosphorylation of AKT and ERK and contributes to the dysregulation of sunitinib in ccRCC cells. Therefore, we identified a novel mechanism for regulating the sensitivity of sunitinib in ccRCC. Therefore, we elucidated a novel mechanism by which RBCK1 regulates sunitinib sensitivity in ccRCC., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells., (© 2022. The Author(s).)

Circular RNAs (circRNAs) play critical roles in clear cell renal cell carcinoma (ccRCC). However, their involvement in sunitinib resistance remains largely unknown. Herein, we identified a novel circRNA, named circME1, which contributes to sunitinib resistance development in ccRCC. CircME1 also promoted proliferation, migration, and invasion of ccRCC cells. Further mechanism analysis showed that circME1 interacted with U1 snRNP at the promoter of its parental gene ME1, thereby upregulating the expression of ME1, enhancing aerobic glycolysis of ccRCC, and promoting its malignant phenotype. Furthermore, ME1 specific inhibitor could effectively repress the oncogenic functions of circME1. Taken together, our study demonstrates that the circME1/ME1 pathway is involved in ccRCC progression and sunitinib resistance development, which may be exploited for anticancer therapy., (© 2022. The Author(s).)

Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active β-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/β-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors (SFRP4, KREMEN1, CXXC4, CSNK1A1 and ZNFR3). Thus, the level of active nuclear β-catenin was increased, leading to increased levels of EMT inducers (SNAI1, SNAI2, ZEB1 and TWIST) and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active β-catenin and EMT inducers., (© 2021. The Author(s).)

Long noncoding RNAs (lncRNAs) have been reported to exert important roles in tumors, including clear cell renal cell carcinoma (ccRCC). PVT1 is an important oncogenic lncRNA which has critical effects on onset and development of various cancers, however, the underlying mechanism of PVT1 functioning in ccRCC remains largely unknown. VHL deficiency-induced HIF2α accumulation is one of the major factors for ccRCC. Here, we identified the potential molecular mechanism of PVT1 in promoting ccRCC development by stabilizing HIF2α. PVT1 was significantly upregulated in ccRCC tissues and high PVT1 expression was associated with poor prognosis of ccRCC patients. Both gain-of-function and loss-of function experiments revealed that PVT1 enhanced ccRCC cells proliferation, migration, and invasion and induced tumor angiogenesis in vitro and in vivo. Mechanistically, PVT1 interacted with HIF2α protein and enhanced its stability by protecting it from ubiquitination-dependent degradation, thereby exerting its biological significance. Meanwhile, HIF2α bound to the enhancer of PVT1 to transactivate its expression. Furthermore, HIF2α specific inhibitor could repress PVT1 expression and its oncogenic functions. Therefore, our study demonstrates that the PVT1/ HIF2α positive feedback loop involves in tumorigenesis and progression of ccRCC, which may be exploited for anticancer therapy., (© 2021. The Author(s).)

The ASPL-TFE3 fusion gene, resulting from t(X;17)(p11.2;q25.3), is one of the most commonly identified fusion genes in Xp11 translocation renal cell carcinoma (tRCC). However, its roles and underlying mechanism in RCC development are not yet clear. Here, we identified ASPL-TFE3 fusion as the most common tRCC subtype in a Chinese population (29/126, 23.03%). This fusion protein translocated into the nucleus and promoted RCC cell proliferation both in vitro and in vivo. Mechanistically, the fusion protein transcriptionally activated the lysosome-autophagy pathway by binding to the promoters of lysosome-related genes. Autophagy, activated by ASPL-TFE3, enabled RCC cells to escape energy stress by promoting the utilization of proteins and lipids. Moreover, we found that the ASPL-TFE3 fusion escaped regulation by the classic mTOR-TFE3 signal and instead activated phospho-mTOR and its downstream targets. Finally, targeting both autophagy and the mTOR axis resulted in a greater antiproliferative effect than single pathway inhibition. In summary, these results confirmed the ASPL-TFE3 fusion as a master regulator of metabolic adaptation mediated by autophagy in tRCC. The simultaneous manipulation of autophagy and the mTOR axis may represent a novel treatment strategy for ASPL-TFE3 fusion RCC.

While the androgen receptor (AR) may influence the progression of clear cell renal cell carcinoma (ccRCC), its role to impact vasculogenic mimicry (VM) to alter the ccRCC progression and metastasis remains obscure. Here, we demonstrated that elevated AR expression was positively correlated with tumor-originated vasculogenesis in ccRCC patients. Consistently, in vitro research revealed AR promoted VM formation in ccRCC cell lines via modulating lncRNA-TANAR/TWIST1 signals. Mechanism dissection showed that AR could increase lncRNA-TANAR (TANAR) expression through binding to the androgen response elements (AREs) located in its promoter region. Moreover, we found that TANAR could impede nonsense-mediated mRNA decay (NMD) of TWIST1 mRNA by direct interaction with TWIST1 5'UTR. A preclinical study using in vivo mouse model with orthotopic xenografts of ccRCC cells further confirmed the in vitro data. Together, these results illustrated that AR-mediated TANAR signals might play a crucial role in ccRCC VM formation and metastasis, and targeting this newly identified AR/TANAR/TWIST1 signaling may help in the development of a novel anti-angiogenesis therapy to better suppress the ccRCC progression.

Recent studies indicated that the androgen receptor (AR) plays important roles in modulating metastasis of VHL-mutant clear cell renal cell carcinoma (ccRCC). However, the precise mechanisms of AR roles in VHL wild-type (VHL-wt) ccRCC, remain unclear. Here we found that AR interacted with VHL to modulate the metastasis of VHL-wt ccRCC via an oxygen-dependent manner. Mechanism dissection revealed that AR could transcriptionally suppress the miR-185-5p expression in the presence of functional VHL-wt protein under a normoxic condition, which might then result in increasing the expression of VEGF-A and VEGF-C via targeting the 3'UTR of mRNAs at a post-transcriptional level. In contrast, under a hypoxic condition, AR could increase miR-185-5p expression to suppress VEGF-C expression, yet this miR-185-5p effect on VEGF-A was reversed via AR's positive regulation on the HIF2α-increased VEGF-A expression that resulted in increasing VEGF-A in the VHL-wt RCC cells. These distinct AR functions under different oxygen conditions may involve the VHL-impacted ubiquitination and nuclear localization of AR. The differential regulation of VEGF-A vs VEGF-C by AR may then result in differential impacts on the ccRCC metastatic destinations of VHL-wt ccRCC cells under different oxygen conditions. These finer mechanisms may help in the development of a novel therapy to better suppress the ccRCC progression under different oxygenization conditions.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer and frequently diagnosed at an advanced stage. It is prone to develop unpredictable metastases even with proper treatment. Antiangiogenic therapy is the most effective medical treatment for metastatic ccRCC. Thus, exploration of novel approaches to inhibit angiogenesis and metastasis may potentially lead to a better therapeutic option for ccRCC. Among all the types of cancer, renal cancer samples exhibited the maximum upregulation of ApoC1 as referred to in the Oncomine database. The expression of ApoC1 was increased accompanied by ccRCC progression. A high level of ApoC1 was closely related to poor survival time in ccRCC patients. Furthermore, ApoC1 was over-expressed in the highly invasive ccRCC cells as compared to that in the low-invasive ccRCC cells. Besides, ApoC1 promoted metastasis of ccRCC cells via EMT pathway, whereas depletion of ApoC1 alleviated these effects. ApoC1 as a novel pro-metastatic factor facilitates the activation of STAT3 and enhances the metastasis of ccRCC cells. Meanwhile, ApoC1 in the exosomes were transferred from the ccRCC cells to the vascular endothelial cells and promoted metastasis of the ccRCC cells via activating STAT3. Finally, the metastatic potential of the ccRCC cells driven by ApoC1 was suppressed by DPP-4 inhibition. Our study not only identifies a novel ApoC1-STAT3 pathway in ccRCC metastasis but also provides direction for the exploration of novel strategies to predict and treat metastatic ccRCC in the future.

PDZK1 downregulation was reported to independently predict poor prognosis of clear cell renal cell carcinoma (ccRCC) patients and induce ccRCC development and progression. However, the underlying mechanism of PDZK1 downregulation remains unknown. Competing endogenous RNA (ceRNA) networks are emerging as new players in gene regulation and are associated with cancer development. ceRNAs regulate other RNA transcripts by competing for shared miRNAs. To investigate the role and mechanism of ceRNAs in PDZK1 downregulation and the development of ccRCC, we searched databases for miRNAs and lncRNAs that regulate PDZK1 expression in ccRCC tissues and assessed their effects in ccRCC. We found that miR-15b was expressed at higher levels in ccRCC tissues, and its upregulation was clinically associated with lower PDZK1 level, larger tumor size and shorter survival time of ccRCC patients. Conversely, a novel lncRNA (lncPENG) was expressed at a lower level in ccRCC tissues, and its downregulation was associated with the same effects as upregulation of miR-15b. Downregulation of miR-15b and upregulation of lncPENG resulted in a significant increase in PDZK1 level and inhibition of proliferation in vitro and in vivo. Mechanistically, lncPENG directly bound to miR-15b and effectively functioned as a sponge for miR-15b to modulate the expression of PDZK1. Thus, lncPENG may function as a ceRNA to attenuate miR-15b-dependent PDZK1 downregulation and inhibit cell proliferation, suggesting that it may be clinically valuable as a therapeutic target and a prognostic biomarker of ccRCC.

Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal human urological malignancies in the world. One of the pathological drivers for ccRCC is the Ras family of small GTPases that function as "molecular switches" in many diseases including ccRCC. Among the GTPases in the Di-Ras family, DIRAS2 gene encodes a GTPase that shares 60% homology to Ras and Rap. Yet little is known about the biological function(s) of Di-Ras2 or how its activities are regulated. In this study, we focused on Di-Ras2, and determined its functions and underlying mechanism during formation of ccRCC. We found that Di-Ras2 was upregulated in ccRCC, and promoted the proliferation, migration and invasion of human ccRCC cells in the absence of von Hippel-Lindau protein (pVHL). Mechanistically, Di-Ras2 induces and regulates ccRCC formation by modulating phosphorylation of the downstream effectors and activating the Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Moreover, Di-Ras2 interacts with E3 ubiquitin ligase, pVHL, which facilitates the ubiquitination and degradation of Di-Ras2. Together, these results indicate a potential function of Di-Ras2 as an oncogene in ccRCC, and these data provide a new perspective of the relationship between pVHL and the MAPK pathway in ccRCC tumorigenesis.

It has been well established that the von Hippel-Lindau/hypoxia-inducible factor α (VHL-HIFα) axis and epidermal growth factor receptor (EGFR) signaling pathway play a critical role in the pathogenesis and progression of renal cell carcinoma (RCC). However, few studies have addressed the relationship between the two oncogenic drivers in RCC. SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase involved in gene transcription and oncogenesis, but its expression and function in RCC remain unclear. In the present study, we found that SMYD3 expression was significantly elevated in RCC tumors and correlated with advanced tumor stage, histological and nuclear grade, and shorter survival. Depletion of SMYD3 inhibited RCC cell proliferation, colony numbers, and xenograft tumor formation, while promoted apoptosis. Mechanistically, SMYD3 cooperates with SP1 to transcriptionally promote EGFR expression, amplifying its downstream signaling activity. TCGA data analyses revealed a significantly increased SMYD3 expression in primary RCC tumors carrying the loss-of-function VHL mutations. We further showed that HIF-2α can directly bind to the SMYD3 promoter and subsequently induced SMYD3 transcription and expression. Taken together, we identify the VHL/HIF-2α/SMYD3 signaling cascade-mediated EGFR hyperactivity through which SMYD3 promotes RCC progression. Our study suggests that SMYD3 is a potential therapeutic target and prognostic factor in RCC.

Renal cell carcinoma (RCC) comprises a diverse group of malignancies arising from the nephron. The most prevalent type, clear cell renal cell carcinoma (ccRCC), is characterized by genetic mutations in factors governing the hypoxia signaling pathway, resulting in metabolic dysregulation, heightened angiogenesis, intratumoral heterogeneity, and deleterious tumor microenvironmental (TME) crosstalk. Identification of specific genetic variances has led to therapeutic innovation and improved survival for patients with ccRCC. Current barriers to effective long-term therapeutic success highlight the need for continued drug development using improved modeling systems. ccRCC preclinical models can be grouped into three broad categories: cell line, mouse, and 3D models. Yet, the breadth of important unanswered questions in ccRCC research far exceeds the accessibility of model systems capable of carrying them out. Accordingly, we review the strengths, weaknesses, and therapeutic implications of each model system that are relied upon today.

Renal cell carcinoma (RCC) is one of the most lethal urological tumors. Using sunitinib to improve the survival has become the first-line therapy for metastatic RCC patients. However, the occurrence of sunitinib resistance in the clinical application has curtailed its efficacy. Here we found TR4 nuclear receptor might alter the sunitinib resistance to RCC via altering the TR4/lncTASR/AXL signaling. Mechanism dissection revealed that TR4 could modulate lncTASR (ENST00000600671.1) expression via transcriptional regulation, which might then increase AXL protein expression via enhancing the stability of AXL mRNA to increase the sunitinib resistance in RCC. Human clinical surveys also linked the expression of TR4, lncTASR, and AXL to the RCC survival, and results from multiple RCC cell lines revealed that targeting this newly identified TR4-mediated signaling with small molecules, including tretinoin, metformin, or TR4-shRNAs, all led to increase the sunitinib sensitivity to better suppress the RCC progression, and our preclinical study using the in vivo mouse model further proved tretinoin had a better synergistic effect to increase sunitinib sensitivity to suppress RCC progression. Future successful clinical trials may help in the development of a novel therapy to better suppress the RCC progression.

miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3'UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.

One-carbon metabolism plays a central role in a broad array of metabolic processes required for the survival and growth of tumor cells. However, the molecular basis of how one-carbon metabolism may influence RNA methylation and tumorigenesis remains largely unknown. Here we show MTHFD2, a mitochondrial enzyme involved in one-carbon metabolism, contributes to the progression of renal cell carcinoma (RCC) via a novel epitranscriptomic mechanism that involves HIF-2α. We found that expression of MTHFD2 was significantly elevated in human RCC tissues, and MTHFD2 knockdown strongly reduced xenograft tumor growth. Mechanistically, using an unbiased methylated RNA immunoprecipitation sequencing (meRIP-Seq) approach, we found that MTHFD2 plays a critical role in controlling global N 6 -methyladenosine (m 6 A) methylation levels, including the m 6 A methylation of HIF-2α mRNA, which results in enhanced translation of HIF-2α. Enhanced HIF-2α translation, in turn, promotes the aerobic glycolysis, linking one-carbon metabolism to HIF-2α-dependent metabolic reprogramming through RNA methylation. Our findings also suggest that MTHFD2 and HIF-2α form a positive feedforward loop in RCC, promoting metabolic reprograming and tumor growth. Taken together, our results suggest that MTHFD2 links RNA methylation status to the metabolic state of tumor cells in RCC.

UHRF1 is an important epigenetic regulator that belongs to the UHRF family. Overexpression of UHRF1 has been found in many kinds of tumors and its overexpression is associated with poor prognosis and short survival in certain cancer types. However, its function in renal cell carcinoma (RCC) is not clear. Here we report that RCC tumor tissues had obviously higher UHRF1 expression than normal renal tissues. Downregulation of UHRF1 by siRNA or shRNA in RCC cell lines resulted in decreased cell viability, inhibited cell migration and invasion, and increased apoptosis. UHRF1 knockdown RCC xenografts also resulted in obviously inhibited tumor growth in vivo. After downregulation of UHRF1 in RCC cells, the expression of TXNIP was upregulated. In addition, after UHRF1 and TXNIP were simultaneously downregulated, cell viability and cell invasion increased, whereas cell apoptosis decreased compared with UHRF1 single downregulated cells. We also showed that UHRF1 could recruit HDAC1 to the TXNIP promoter and mediate the deacetylation of histone H3K9, resulting in the inhibition of TXNIP expression. Our results confirm that UHRF1 has oncogenic function in RCC and UHRF1 may promote tumor progression through epigenetic regulation of TXNIP. UHRF1 might be used as a therapeutic target for RCC treatment.

CXC chemokine receptor 4 (CXCR4) has been suggested to play a critical role in cancer metastasis. Some studies have described CXCR4 nuclear localization in metastatic lesions of renal cell carcinoma (RCC), which has been suggested to be correlated with cancer metastasis. However, the underlying mechanism and clinical significance of CXCR4 nuclear localization remains unknown. Here, we show that CXCR4 nuclear localization is more likely to occur in RCC tissues, especially in metastases, and is associated with poor prognosis. CXCR4 nuclear localization requires its nuclear localization sequence (NLS, residues 146-RPRK-149). After the mutation of NLS in CXCR4, CXCR4 nuclear localization in RCC cells is lost. Nuclear localization of CXCR4 promoted RCC tumorigenicity both in vitro and in vivo. Mechanistically, we found that CXCR4 and hypoxia-inducible factor-1α (HIF-1α) colocalized in RCC cells and interacted with each other. Moreover, CXCR4 nuclear localization promoted nuclear accumulation of HIF-1α, thereby promoting the expression of genes downstream of HIF-1α. Reciprocally, nuclear HIF-1α promoted CXCR4 transcription, thus forming a feed-forward loop. Subcellular CXCR4 and HIF-1α expression levels were independent adverse prognostic factors and could be combined with TNM stage to generate a predictive nomogram of the clinical outcome of patients with RCC. Therefore, our findings indicate that CXCR4 nuclear translocation plays a critical role in RCC metastasis and may serve as a prognostic biomarker and potential therapeutic target.

The von Hippel-Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previously we reported, using in silico and in vitro analyses, that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1α. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. Here we examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome.

Metastatic clear cell renal cell carcinoma (CCC) remains incurable despite advances in the development of anti-angiogenic targeted therapies and the emergence of immune checkpoint inhibitors. We have previously shown that the sonic hedgehog-Gli signaling pathway is oncogenic in CCC allowing us to identify the developmental Lim1 transcription factor as a Gli target and as a new oncogene in CCC regulating cell proliferation and apoptosis, and promoting tumor growth. In this previous study, preliminary in vitro results also suggested that Lim1 may be implicated in metastatic spread. Here we investigated the potential pro-metastatic role of Lim1 in advanced CCC (1) in vitro using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene either naturally or by gene transfer and (2) ex vivo in 30 CCC metastatic tissues, including lymph nodes, lung, skin, bone, and adrenal metastases, and (3) in vivo, using a metastatic model by intravenous injection of siRNA-transfected cells into Balb/c nude. Our in vitro results reveal that Lim1 knockdown time-dependently decreased CCC cell motility, migration, invasion, and clonogenicity by up to 50% regardless of their VHL status. Investigating the molecular machinery involved in these processes, we identified a large panel of Lim1 targets known to be involved in cell adhesion (paxillin and fibronectin), epithelial-mesenchymal transition (Twist1/2 and snail), invasion (MMP1/2/3/8/9), and metastatic progression (CXCR4, SDF-1, and ANG-1). Importantly, Lim1 was found constitutively expressed in all metastatic tissues. The H-score in metastatic tissues being significantly superior to the score in the corresponding primary tumor tissues (P value = 0.009). Furthermore, we showed that Lim1 silencing decreases pulmonary metastasis development in terms of number and size in the in vivo metastatic model of human CCC. Taken together, these experiments strengthen the potential therapeutic value of Lim1 targeting as a promising novel approach for treating metastatic human CCC.

While TR4 nuclear receptor plays key roles to promote prostate cancer progression, its roles to alter the progression of clear cell renal cell carcinoma (ccRCC), remains unclear. Here, we demonstrate that TR4 can promote the ccRCC cell vasculogenic mimicry (VM) formation and its associated metastasis via modulating the miR490-3p/vimentin (VIM) signals. Mechanism dissection revealed that TR4 might increase the oncogene VIM expression via decreasing the miR-490-3p expression through direct binding to the TR4-response-elements (TR4REs) on the promoter region of miR-490-3p, which might then directly target the 3' UTR of VIM-mRNA to increase its protein expression. Preclinical studies using the in vivo mouse model with xenografted RCC Caki-1 cells into the sub-renal capsule of nude mice also found that TR4 could promote the ccRCC VM and its associated metastasis via modulating the miR490-3p/VIM signals. Together, results from preclinical studies using multiple RCC cell lines and the in vivo mouse model all conclude that TR4 may play a key role to promote ccRCC VM formation and metastasis and targeting the newly identified TR4/miR-490-3p/VIM signals with small molecules may help us to develop a new therapeutic approach to better suppress the ccRCC metastasis.

Recent studies indicated that the estrogen receptor beta (ERβ) could affect the progression of prostate and bladder tumors, however, its roles in the renal cell carcinoma (RCC), remain to be elucidated. Here, we provide clinical evidence that ERβ expression is correlated in a negative manner with the overall survival/disease-free survival in RCC patients. Mechanism dissection revealed that targeting ERβ with ERβ-shRNA and stimulating the transactivation of ERβ with 17β-estradiol or environmental endocrine disrupting chemicals, all resulted in altering the lncRNA HOTAIR expression. The ERβ-modulated HOTAIR is able to function via antagonizing several microRNAs, including miR-138, miR-200c, miR-204, or miR-217 to impact various oncogenes, including ADAM9, CCND2, EZH2, VEGFA, VIM, ZEB1, and ZEB2, to promote RCC proliferation and invasion. Together, the identification of the ERβ-HOTAIR axis may provide us new biomarkers and/or therapeutic targets to better suppress RCC progression in the future.

TGF-β regulates both the tumor-forming and migratory abilities of various types of cancer cells. However, it is unclear how the loss of TGF-β signaling components affects these abilities in clear-cell renal cell carcinoma (ccRCC). In this study, we investigated the role of TGFBR3 (TGF-β type III receptor, also known as betaglycan) in ccRCC. Database analysis revealed decreased expression of TGFBR3 in ccRCC tissues, which correlated with poor prognosis in patients. Orthotopic inoculation experiments using immunocompromised mice indicated that low TGFBR3 expression in ccRCC cells enhanced primary tumor formation and lung metastasis. In the presence of TGFBR3, TGF-β2 decreased the aldehyde dehydrogenase (ALDH)-positive ccRCC cell population, in which renal cancer-initiating cells are enriched. Loss of TGFBR3 also enhanced cell migration in cell culture and induced expression of several mesenchymal markers in a TGF-β-independent manner. Increased lamellipodium formation by FAK-PI3K signaling was observed with TGFBR3 downregulation, and this contributed to TGF-β-independent cell migration in ccRCC cells. Taken together, our findings reveal that loss of TGFBR3 endows ccRCC cells with multiple metastatic abilities through TGF-β-dependent and independent pathways.

Mutations in VHL underlie von Hippel-Lindau (VHL) disease, a hereditary cancer syndrome with several subtypes depending on the risk of developing certain combination of classic features, such as clear cell renal cell carcinoma (ccRCC), hemangioblastoma and pheochromocytoma. Although numerous potential substrates and functions of pVHL have been described over the past decade, the best-defined role of pVHL has remained as the negative regulator of the heterodimeric hypoxia-inducible factor (HIF) transcription factor via the oxygen-dependent ubiquitin-mediated degradation of HIF-α subunit. Despite the seminal discoveries that led to the molecular elucidation of the mammalian oxygen-sensing VHL-HIF axis, which have provided several rational therapies, the mechanisms underlying the complex genotype-phenotype correlation in VHL disease are unclear. This review will discuss and highlight the studies that have provided interesting insights as well as uncertainties to the underlying mechanisms governing VHL disease.

Renal cell carcinoma (RCC) is one of the most aggressive urologic cancers, however, the mechanism on supporting RCC carcinogenesis is still not clear. By using gene expression profile analysis and functional clustering, PDZ domain-containing 1 (PDZK1) was revealed to be downregulated in human clear cell renal cell carcinoma (ccRCC) samples, which was also verified in several independent public ccRCC data sets. Using PDZK1 overexpression and knockdown models in ccRCC cell lines, we demonstrated that PDZK1 inhibited cell proliferation, cell cycle G1/S phase transition, cell migration and invasion, indicating a tumor-suppressor role in the development and progression of ccRCC. Our study further demonstrated that PDZK1 inhibited cell proliferation and migration of ccRCC via targeting SHP-1. PDZK1 was further identified to suppress cell proliferation by blocking SHP-1 phosphorylation at Tyr536 via inhibition of the association between SHP-1 and PLCβ3, and then retarding Akt phosphorylation and promoting STAT5 phosphorylation in ccRCC cells. Moreover, the inhibitive effects of PDZK1 on SHP-1 phosphorylation and the tumor growth were verified in vivo by xenograft tumor studies. Accordingly, PDZK1 expression was negatively correlated with SHP-1 activation and phosphorylation, advanced pathologic stage, tumor weight and size, and prognosis of ccRCC patients. These findings have provided first lines of evidences that PDZK1 expression is negatively correlated with SHP-1 activation and poor clinical outcomes in ccRCC. PDZK1 was identified as a novel tumor suppressor in ccRCC by negating SHP-1 activity.

The hypoxia-regulated tumor-suppressor von Hippel-Lindau (VHL) is an E3 ligase that recognizes its substrates as part of an oxygen-dependent prolyl hydroxylase (PHD) reaction, with hypoxia-inducible factor α (HIFα) being its most notable substrate. Here we report that VHL has an equally important function distinct from its hypoxia-regulated activity. We find that Aurora kinase A (AURKA) is a novel, hypoxia-independent target for VHL ubiquitination. In contrast to its hypoxia-regulated activity, VHL mono-, rather than poly-ubiquitinates AURKA, in a PHD-independent reaction targeting AURKA for degradation in quiescent cells, where degradation of AURKA is required to maintain the primary cilium. Tumor-associated variants of VHL differentiate between these two functions, as a pathogenic VHL mutant that retains intrinsic ability to ubiquitinate HIFα is unable to ubiquitinate AURKA. Together, these data identify VHL as an E3 ligase with important cellular functions under both normoxic and hypoxic conditions.

Although the use of sorafenib appears to increase the survival rate of renal cell carcinoma (RCC) patients, there is also a proportion of patients who exhibit a poor primary response to sorafenib therapy. It is therefore critical to elucidate the mechanisms underlying sorafenib resistance and find representative biomarkers for sorafenib treatment in RCC patients. Herein, we identified a long non-coding RNA referred to as lncRNA-SRLR (sorafenib resistance-associated lncRNA in RCC) that is upregulated in intrinsically sorafenib-resistant RCCs. lncRNA-SRLR knockdown sensitized nonresponsive RCC cells to sorafenib treatment, whereas the overexpression of lncRNA-SRLR conferred sorafenib resistance to responsive RCC cells. Mechanistically, lncRNA-SRLR directly binds to NF-κB and promotes IL-6 transcription, leading to the activation of STAT3 and the development of sorafenib tolerance. A STAT3 inhibitor and IL-6-receptor antagonist both restored the response to sorafenib treatment. Moreover, a clinical investigation demonstrated that high levels of lncRNA-SRLR correlated with poor responses to sorafenib therapy in RCC patients. Collectively, lncRNA-SRLR may serve as not only a predictive biomarker for inherent sorafenib resistance but also as a therapeutic target to enhance responses to sorafenib in RCC patients.

Clear cell renal cell carcinoma (CC-RCC) is the most lethal of all genitourinary cancers. The functional loss of the von Hippel-Lindau (VHL) gene occurs in 90% of CC-RCC, driving cancer progression. The objective of this study was to identify chemical compounds that are synthetically lethal with VHL deficiency in CC-RCC. An annotated chemical library, the library of pharmacologically active compounds (LOPAC), was screened in parallel on VHL-deficient RCC4 cells and RCC4VHL cells with re-introduced VHL. The ROCK inhibitor, Y-27632, was identified and validated for selective targeting of VHL-deficient CC-RCC in multiple genetic backgrounds by clonogenic assays. Downregulation of ROCK1 by small interfering RNA (siRNA) selectively reduced the colony-forming ability of VHL-deficient CC-RCC, thus mimicking the effect of Y-27632 treatment, whereas downregulation of ROCK2 had no effect. In addition, two other ROCK inhibitors, RKI 1447 and GSK 429286, selectively targeted VHL-deficient CC-RCC. CC-RCC treatment with ROCK inhibitors is cytotoxic and cytostatic based on bromodeoxyuridine (BrdU) assay, propidium iodide (PI) staining and growth curves, and blocks cell migration based on transwell assay. On the one hand, knockdown of hypoxia-inducible factor (HIF) β in the VHL-deficient CC-RCC had a protective effect against Y-27632 treatment, mimicking VHL reintroduction. On the other hand, CC-RCCVHL cells were sensitized to Y-27632 treatment in hypoxia (2% O 2 ). These results suggest that synthetic lethality between ROCK inhibition and VHL deficiency is dependent on HIF activation. Moreover, HIF1α or HIF2α overexpression in CC-RCCVHL cells is sufficient to sensitize them to ROCK inhibition. Finally, Y-27632 treatment inhibited growth of subcutaneous 786-OT1 CC-RCC tumors in mice. Thus, ROCK inhibitors represent potential therapeutics for VHL-deficient CC-RCC.

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long-form variant of VHL was often detected as multiple bands by western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetical and pharmacological approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein and thereby promoted HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases., Competing Interests: The authors declare no conflict of interest.

It is well established that hypoxia contributes to tumor progression in a hypoxia inducible factor-2α (HIF-2α)-dependent manner in renal cell carcinoma (RCC), yet the role of long noncoding RNAs (LncRNAs) involved in hypoxia-mediated RCC progression remains unclear. Here we demonstrate that LncRNA-SARCC (Suppressing Androgen Receptor in Renal Cell Carcinoma) is differentially regulated by hypoxia in a von Hippel-Lindau (VHL)-dependent manner both in RCC cell culture and clinical specimens. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells. Mechanism dissection reveals that LncRNA-SARCC can post-transcriptionally regulate androgen receptor (AR) by physically binding and destablizing AR protein to suppress AR/HIF-2α/C-MYC signals. In return, HIF-2α can transcriptionally regulate the LncRNA-SARCC expression via binding to hypoxia-responsive elements on the promoter of LncRNA-SARCC. The negative feedback modulation between LncRNA-SARCC/AR complex and HIF-2α signaling may then lead to differentially modulated RCC progression in a VHL-dependent manner. Together, these results may provide us a new therapeutic approach via targeting this newly identified signal from LncRNA-SARCC to AR-mediated HIF-2α/C-MYC signals against RCC progression.

Targeted therapies using small-molecule inhibitors (SMIs) are commonly used in metastatic renal cell cancer (mRCC) patients; patients often develop drug resistance and eventually succumb to disease. Currently, understanding of mechanisms leading to SMIs resistance and any identifiable predictive marker(s) are still lacking. We discovered that DAB2IP, a novel Ras-GTPase-activating protein, was frequently epigenetically silenced in RCC, and DAB2IP loss was correlated with the overall survival of RCC patients. Loss of DAB2IP in RCC cells enhances their sensitivities to growth factor stimulation and resistances to SMI (such as mammalian target of rapamycin (mTOR) inhibitors). Mechanistically, loss of DAB2IP results in the activation of extracellular signal-regulated kinase/RSK1 and phosphoinositide-3 kinase/mTOR pathway, which synergizes the induction of hypoxia-inducible factor (HIF)-2α expression. Consequently, elevated HIF-2α suppresses p21/WAF1 expression that is associated with resistance to mTOR inhibitors. Thus combinatorial targeting both pathways resulted in a synergistic tumor inhibition. DAB2IP appears to be a new prognostic/predictive marker for mRCC patients, and its function provides a new insight into the molecular mechanisms of drug resistance to mTOR inhibitors, which also can be used to develop new strategies to overcome drug-resistant mRCC.

The multifunctional enzyme transglutaminase 2 (TG2) primarily catalyzes cross-linking reactions of proteins via (γ-glutamyl) lysine bonds. Several recent findings indicate that altered regulation of intracellular TG2 levels affects renal cancer. Elevated TG2 expression is observed in renal cancer. However, the molecular mechanism underlying TG2 degradation is not completely understood. Carboxyl-terminus of Hsp70-interacting protein (CHIP) functions as an ubiquitin E3 ligase. Previous studies reveal that CHIP deficiency mice displayed a reduced life span with accelerated aging in kidney tissues. Here we show that CHIP promotes polyubiquitination of TG2 and its subsequent proteasomal degradation. In addition, TG2 upregulation contributes to enhanced kidney tumorigenesis. Furthermore, CHIP-mediated TG2 downregulation is critical for the suppression of kidney tumor growth and angiogenesis. Notably, our findings are further supported by decreased CHIP expression in human renal cancer tissues and renal cancer cells. The present work reveals that CHIP-mediated TG2 ubiquitination and proteasomal degradation represent a novel regulatory mechanism that controls intracellular TG2 levels. Alterations in this pathway result in TG2 hyperexpression and consequently contribute to renal cancer.

Tumor-specific alternative splicing is implicated in the progression of cancer, including clear-cell renal cell carcinoma (ccRCC). Using ccRCC RNA sequencing data from The Cancer Genome Atlas, we found that epithelial splicing regulatory protein 2 (ESRP2), one of the key regulators of alternative splicing in epithelial cells, is expressed in ccRCC. ESRP2 mRNA expression did not correlate with the overall survival rate of ccRCC patients, but the expression of some ESRP-target exons correlated with the good prognosis and with the expression of Arkadia (also known as RNF111) in ccRCC. Arkadia physically interacted with ESRP2, induced polyubiquitination and modulated its splicing function. Arkadia and ESRP2 suppressed ccRCC tumor growth in a coordinated manner. Lower expression of Arkadia correlated with advanced tumor stages and poor outcomes in ccRCC patients. This study thus reveals a novel tumor-suppressive role of the Arkadia-ESRP2 axis in ccRCC.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor drives the development of clear-cell renal cell carcinoma (ccRCC) through hypoxia-inducible factors (HIFs). Although ccRCC cells exhibit constitutive normoxic HIF signaling, the potential role of hypoxia in this setting is not fully understood. We show here that the ccRCC cell lines RCC4 and RCC10, which express mutant versions of VHL, have reduced HIF1α expression in hypoxia, whereas HIF2α expression is increased or not affected. Similar findings were observed in normoxia after abrogation of prolyl hydroxylase activity by siRNA or pharmacological inhibition, and by siRNA inhibition of mutant VHL. This reduction of HIF1α protein is due to proteasome-dependent degradation and is mediated by the E3 ubiquitin ligase SART1. HIF1α degradation favors ccRCC proliferation, in line with the previously recognized tumor suppressor capability of HIF1α. Our data indicate that mutant VHL can protect HIF1α from SART1-dependent degradation in normoxic conditions, but this protection is lost in hypoxic settings, favoring hypoxia-dependent ccRCC proliferation. This mechanism of HIF1α degradation might operate in some VHL-related clear-cell renal carcinomas in which the deletion of HIF1α locus does not occur.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Mutations of the tumor suppressor gene von Hippel-Lindau (VHL) can lead to benign and malignant tumors, including clear-cell renal cell carcinoma (ccRCC). To understand the progression of ccRCC, we generated a novel mouse Vhlh conditional knockout, using Hoxb7-driven Cre that is specific for the collecting ducts and a subset of distal tubules. These mice exhibited wide-spread epithelial disruption and interstitial inflammation as early as 2 months of age with high penetrance. Lesions are cystic, show severe fibrosis and display significant hyperplasia. An abundance of infiltrating macrophages and lymphocytes was detected. Interestingly, the Vhlh mutant lesions could be rescued when Hif-1α, but not Hif-2α, was also knocked out. In addition, administration of a JAK1/2 kinase inhibitor alleviated the Vhlh knockout phenotypes. Taken together, these results suggest that HIF-1α-dependent inflammation and fibrosis may be an early event in the development of ccRCC.

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

Functional loss of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL), which is part of an E3-ubiquitin ligase complex, initiates most inherited and sporadic clear-cell renal cell carcinomas (ccRCC). Genetic inactivation of the TP53 gene in ccRCC is rare, suggesting that an alternate mechanism alleviates the selective pressure for TP53 mutations in ccRCC. Here we use a zebrafish model to describe the functional consequences of pVHL loss on the p53/Mdm2 pathway. We show that p53 is stabilized in the absence of pVHL and becomes hyperstabilized upon DNA damage, which we propose is because of a novel in vivo interaction revealed between human pVHL and a negative regulator of Mdm2, the programmed cell death 5 (PDCD5) protein. PDCD5 is normally localized at the plasma membrane and in the cytoplasm. However, upon hypoxia or loss of pVHL, PDCD5 relocalizes to the nucleus, an event that is coupled to the degradation of Mdm2. Despite the subsequent hyperstabilization and normal transcriptional activity of p53, we find that zebrafish vhl(-/-) cells are still as highly resistant to DNA damage-induced cell cycle arrest and apoptosis as human ccRCC cells. We suggest this is because of a marked increase in expression of birc5a, the zebrafish homolog of Survivin. Accordingly, when we knock down Survivin in human ccRCC cells we are able to restore caspase activity in response to DNA damage. Taken together, our study describes a new mechanism for p53 stabilization through PDCD5 upon hypoxia or pVHL loss, and reveals new clinical potential for the treatment of pathobiological disorders linked to hypoxic stress.

Recent studies have demonstrated that in clear cell renal cell carcinoma (ccRCC) several chromatin remodeling enzymes are genetically inactivated. Although, growing evidence in cancer models has demonstrated the importance of epigenetic changes, currently only changes in DNA methylation can be accurately determined from clinical samples. To address this limitation, we have applied formaldehyde-assisted isolation of regulatory elements (FAIREs) combined with next-generation sequencing (FAIRE-seq) to identify specific changes in chromatin accessibility in clinical samples of ccRCC. We modified the FAIRE procedure to allow us to examine chromatin accessibility for small samples of solid tumors. Our FAIRE results were compared with DNA-methylation analysis and show how chromatin accessibility decreases at many sites where DNA-methylation remains unchanged. In addition, our FAIRE-seq analysis allowed us to identify regulatory elements associated with both normal and tumor tissue. We have identified decreases in chromatin accessibility at key ccRCC-linked genes, including PBRM1, SETD2 and MLL2. Overall, our results demonstrate the power of examining multiple aspects of the epigenome.

Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The 'signal transducer and activator of transcription-3' (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.

Birt-Hogg-Dubé (BHD) syndrome, is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild-type allele consistent with a tumor suppressor gene function. In a search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screening approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and previously implicated in actin reorganization) specifically induced Caspase3/7 activity in a dose-dependent manner (up to six-fold increase, 10 nM, 72 h) in two human FLCN-deficient cell lines (BHD-origin renal cell carcinoma UOK257 and thyroid carcinoma FTC133) but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was accompanied by increased expression of SSH1 and SSH3 (suggesting a compensatory regulatory mechanism among members of SSH family). FLCN-null cells exhibited evidence of dysregulated cofilin de/phosphorylation pathways. Knockdown of SSH2 in FLCN-null cells was associated with an alteration in cell cycle kinetics (20% increase in G1, 30% and 40% decrease in S and G2M, respectively). Combination treatment of multiple SSH family (SSH2 plus SSH1 and/or SSH3) siRNAs potentiated induction of Caspase3/7 activity and changes in the cell cycle kinetics. These data indicate that: (a) apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through cell cycle arrest; (b) SSH2 represents a potential therapeutic target for the development of agents for the treatment of BHD syndrome and, possibly, related tumors.

Non-neuronal expression of components of the glutamatergic system has been increasingly observed, and our laboratory previously had demonstrated the etiological role of ectopically expressed metabotropic glutamate receptor 1 (Grm1/mGluR1) in mouse models of melanoma. We hypothesize that inappropriate glutamatergic signaling in other cell types can dysregulate growth leading to transformation and tumorigenesis. As most cancers are carcinomas, we selected an immortalized primary baby mouse kidney (iBMK) cell model to assess whether Grm1 can transform epithelial cells. These iBMK cells, engineered to be immortal yet nontumorigenic and retaining normal epithelial characteristics, were used as recipients for exogenous Grm1 cDNA. Several stable Grm1-expressing clones were isolated and the Grm1-receptors were shown to be functional, as evidenced by the accumulation of second messengers in response to Grm1 agonist. Additionally activated by agonist were mitogen-activated protein kinase (MAPK) and AKT/protein kinase B signaling cascades, the major intracellular pathways shown by many investigators to be critical in melanomagenesis and other neoplasms. These Grm1-iBMK cells exhibited enhanced cell proliferation in in vitro methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays and significant tumorigenicity in in vivo allografts. Persistent Grm1 expression was required for the maintenance of the in vivo tumorigenic phenotype as demonstrated by an inducible Grm1-silencing RNA. These are the first results that indicate that Grm1 can be an oncogene in epithelial cells. In addition, relevance to human disease in the corresponding tumor type of renal cell carcinoma (RCC) may be suggested by observed expression of GRM1/mGluR1 in a number of RCC tumor biopsy samples and cell lines, and the effects of GRM1 modulation on tumorigenicity therein. Moreover, RCC cell lines exhibited elevated levels of extracellular glutamate, and some lines responded to drugs, which modulate the glutamatergic system. These findings imply a possible role for glutamate signaling apparatus in RCC cell growth, and that the glutamatergic system may be a therapeutic target in RCC.

Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

Incidence of kidney cancer is on the rise, and a better understanding of molecular mechanisms involved in the cancer invasion and metastasis is required for the development of curative therapeutics. In this study, we report that the proinflammatory cytokine prostaglandin E2 (PGE2) induces the malignant SN12C, but not benign HK2 kidney cell invasion. The PGE2 increases SN12C cell invasion through a signal pathway that encompasses EP2 and EP4, Akt, small GTPase RalA and Ral·GTP inactivator RGC2. The results support the idea that targeted interference of EP2/EP4 signal to RalA·GTP may provide benefit to patients diagnosed with advanced kidney cancer.

In clear-cell renal cell carcinoma (ccRCC), inactivation of the tumor suppressor von Hippel-Lindau (VHL) occurs in the majority of the tumors and is causal for the pathogenesis of ccRCC. Recently, a large-scale genomic sequencing study of ccRCC tumors revealed that enzymes that regulate histone H3 lysine 4 trimethylation (H3K4Me3), such as JARID1C/KDM5C/SMCX and MLL2, were mutated in ccRCC tumors, suggesting that H3K4Me3 might have an important role in regulating gene expression and tumorigenesis. In this study we report that in VHL-deficient ccRCC cells, the overall H3K4Me3 levels were significantly lower than that of VHL+/+ counterparts. Furthermore, this was hypoxia-inducible factor (HIF) dependent, as depletion of HIF subunits by small hairpin RNA in VHL-deficient ccRCC cells restored H3K4Me3 levels. In addition, we demonstrated that only loss of JARID1C, not JARID1A or JARID1B, abolished the difference of H3K4Me3 levels between VHL-/- and VHL+/+ cells, and JARID1C displayed HIF-dependent expression pattern. JARID1C in VHL-/- cells was responsible for the suppression of HIF-responsive genes insulin-like growth factor-binding protein 3 (IGFBP3), DNAJC12, COL6A1, growth and differentiation factor 15 (GDF15) and density-enhanced phosphatase 1. Consistent with these findings, the H3K4Me3 levels at the promoters of IGFBP3, DNAJC12, COL6A1 and GDF15 were lower in VHL-/- cells than in VHL+/+ cells, and the differences disappeared after JARID1C depletion. Although HIF2α is an oncogene in ccRCC, some of its targets might have tumor suppressive activity. Consistent with this, knockdown of JARID1C in 786-O VHL-/- ccRCC cells significantly enhanced tumor growth in a xenograft model, suggesting that JARID1C is tumor suppressive and its mutations are tumor promoting in ccRCC. Thus, VHL inactivation decreases H3K4Me3 levels through JARID1C, which alters gene expression and suppresses tumor growth.

The tumor suppressor gene von Hippel-Lindau (VHL) is involved in the development of sporadic clear-cell renal cell carcinoma (RCC). VHL interferes with angiogenesis and also controls cell adhesion and invasion. Therapies that target VHL-controlled genes are currently being evaluated in RCC patients. RCC is a immunogenic tumor and treatment with interleukin-2 (IL2) or interferon (IFN)-α results in regression in some patients. We used two renal tumor cell lines (RCC6 and RCC4) carrying VHL loss-of-function mutations to investigate the role of mutant VHL in susceptibility to natural killer (NK) cell-mediated lysis. The RCC6 and RCC4 cell lines were transfected with the wild-type gene to restore the function of VHL. The presence of the gene in RCC cells downregulated hypoxia-inducible factor (HIF)-1α and subsequently decreased vascular endothelial growth factor (VEGF) production. Relative to control transfectants and parental cells, pVHL-transfected cell lines activated resting and IL2-activated NK cells less strongly, as assessed by IFNγ secretion, NK degranulation and cell lysis. NKG2A, a human leukocyte antigen (HLA)-I-specific inhibitory NK receptor, controls the lysis of tumor targets. We show that HLA-I expression in RCC-pVHL cells is stronger than that in parental and controls cells, although the expression of activating receptor NK ligands remains unchanged. Blocking NKG2A/HLA-I interactions substantially increased lysis of RCC-pVHL, but had little effect on the lysis of VHL-mutated RCC cell lines. In addition, in response to IFNα, the exponential growth of RCC-pVHL was inhibited more than that of RCC-pE cells, indicating that VHL mutations may be involved in IFNα resistance. These results indicate that a decreased expression of HLA-I molecules in mutated VHL renal tumor cells sensitizes them to NK-mediated lysis. These results suggest that combined immunotherapy with anti-angiogenic drugs may be beneficial for patients with mutated VHL.