Lijun Zhou

Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma.

Antiangiogenic therapy resistance occurs frequently in patients with metastatic renal cell carcinoma (RCC). The purpose of this study was to understand the mechanism of resistance to sunitinib, an antiangiogenic small molecule, and to exploit this mechanism therapeutically. We hypothesized that sunitinib-induced upregulation of the prometastatic MET and AXL receptors is associated with resistance to sunitinib and with more aggressive tumor behavior. In the present study, tissue microarrays containing sunitinib-treated and untreated RCC tissues were stained with MET and AXL antibodies. The low malignant RCC cell line 786-O was chronically treated with sunitinib and assayed for AXL, MET, epithelial–mesenchymal transition (EMT) protein expression and activation. Co-culture experiments were used to examine the effect of sunitinib pretreatment on endothelial cell growth. The effects of AXL and MET were evaluated in various cell-based models by short hairpin RNA or inhibition by cabozantinib, the multi-tyrosine kinases inhibitor that targets vascular endothelial growth factor receptor, MET and AXL. Xenograft mouse models tested the ability of cabozantinib to rescue sunitinib resistance. We demonstrated that increased AXL and MET expression was associated with inferior clinical outcome in patients. Chronic sunitinib treatment of RCC cell lines activated both AXL and MET, induced EMT-associated gene expression changes, including upregulation of Snail and β-catenin, and increased cell migration and invasion. Pretreatment with sunitinib enhanced angiogenesis in 786-0/human umbilical vein endothelial cell co-culture models. The suppression of AXL or MET expression and the inhibition of AXL and MET activation using cabozantinib both impaired chronic sunitinib treatment-induced prometastatic behavior in cell culture and rescued acquired resistance to sunitinib in xenograft models. In summary, chronic sunitinib treatment induces the activation of AXL and MET signaling and promotes prometastatic behavior and angiogenesis. The inhibition of AXL and MET activity may overcome resistance induced by prolonged sunitinib therapy in metastatic RCC.

Resistance to Antiangiogenic Therapy Is Associated with an Immunosuppressive Tumor Microenvironment in Metastatic Renal Cell Carcinoma

Therapeutic PD-1/PD-L1 blockade requires preexisting tumor-infiltrating T cells. In a subset of metastatic RCC patients, antiangiogenic therapy increased T-cell infiltration and PD-L1 upregulation, increasing the likelihood that they may uniquely benefit from combination checkpoint and antiangiogenic therapy. Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer, and antiangiogenic therapy is the current mainstay of treatment. Patients with RCC develop innate or adaptive resistance to antiangiogenic therapy. There is a need to identify biomarkers that predict therapeutic resistance and guide combination therapy. We assessed the interaction between antiangiogenic therapy and the tumor immune microenvironment and determined their impact on clinical outcome. We found that antiangiogenic therapy–treated RCC primary tumors showed increased infiltration of CD4+ and CD8+ T lymphocytes, which was inversely related to patient overall survival and progression-free survival. Furthermore, specimens from patients treated with antiangiogenic therapy showed higher infiltration of CD4+FOXP3+ regulatory T cells and enhanced expression of checkpoint ligand programed death-ligand 1 (PD-L1). Both immunosuppressive features were correlated with T-lymphocyte infiltration and were negatively related to patient survival. Treatment of RCC cell lines and RCC xenografts in immunodeficient mice with sunitinib also increased tumor PD-L1 expression. Results from this study indicate that antiangiogenic treatment may both positively and negatively regulate the tumor immune microenvironment. These findings generate hypotheses on resistance mechanisms to antiangiogenic therapy and will guide the development of combination therapy with PD-1/PD-L1–blocking agents. Cancer Immunol Res; 3(9); 1017–29. ©2015 AACR.

Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis

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.

Genetic and Pharmacological Strategies to Refunctionalize the von Hippel Lindau R167Q Mutant Protein

Aberrant von Hippel Lindau (VHL) protein function is the underlying driver of VHL-related diseases, including both sporadic and inherited clear cell renal cell carcinoma (ccRCC). About one third of VHL mutations are missense point mutations, with R167Q being the most common VHL point mutation in hereditary VHL disease. Although it has been studied extensively, the ability of VHL-R167Q to downregulate hypoxia-inducible factor 2α (HIF2α) is still controversial. In addition, the manner in which the mutation contributes to tumorigenesis is not fully understood. No therapeutic approach is available to target VHL-R167Q and similar missense point mutations. We analyzed VHL-R167Q proteostasis and function at normoxia, at hypoxia with different oxygen pressure, and in a xenograft mouse model. We showed that the protein levels of VHL-R167Q dictate its ability to downregulate HIF2α and suppress tumor growth. Strikingly, the proteasome inhibitors bortezomib and carfilzomib, which are currently in clinical use, stabilize VHL-R167Q and increase its ability to downregulate HIF2α. VHL-R167Q binds elongin C and elongin B with considerably less avidity than wild-type VHL does but retains residual capacity to generate a VHL-elongin C-elongin B complex, downregulate HIF2α, and suppress tumorigenesis, which could be rescued by increase of VHL-R167Q levels. Finally, we used in silico approaches and identified other missense VHL mutants in addition to VHL-R167Q that might be rescued by similar strategies. Thus, our studies revealed detailed information describing how VHL-R167Q contributes to tumorigenesis and identified a potential targeted therapy for ccRCC and other VHL-related disease in patients carrying VHL-R167Q or similar missense mutations.

HNF1B loss exacerbates the development of chromophobe renal cell carcinomas

Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313-26. ©2017 AACR.

Abstract 687: The mechanisms of anti-VEGF therapy resistance in renal cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Anti-VEGF therapy is a mainstay of treatment for metastatic renal cell carcinoma (RCC). Treatment is either via targeting of the ligand itself, or via inhibition of the receptor with small molecule VEGF receptor inhibitors like sunitinib, pazopanib or axitinib. Unfortunately, between 10 and 20 percent of patients have tumors that are refractory to anti-VEGF therapy, and the majority of remaining patients eventually demonstrate tumor growth after a median of 10-12 months of therapy. Thus, an important goal is to develop strategies to overcome anti-VEGF therapy resistance. We hypothesized that the receptor tyrosine kinases AXL and MET, which are implicated in oncogenesis and the development of resistance to targeted therapies in some cancers, are involved in anti-VEGF therapy resistance in RCC, and this resistance can be overcome with an inhibitor of AXL and MET. In the current study, we investigated both tumor autonomous and endothelial-specific drivers of anti-VEGF/sunitinib therapy resistance, bearing in mind that the primary presumed target of sunitinib in RCC is the endothelial cell. We observed that sunitinib treatment stimulated both AXL expression and kinase activation in the 786-0 RCC cell line. Chronic sunitinib treatment (four weeks, 5µM) also elevated MET activity, and stimulated the activation of key effector kinases, AKT and ERK. In addition, the ability of sunitinib to inhibit cell proliferation was lost in786-O cells chronically exposed to sunitinib. Cell migration in vitro was inhibited when AXL and MET expression was suppressed by shRNA knockdown. We also observed that cabozantinib, a novel MET/VEGFR/AXL inhibitor, effectively suppressed the activation of MET, AXL, AKT and ERK both in parental and sunitinib-resistant 786-O cells, and inhibited cell migration in vitro. Angiogenesis is critical for RCC tumor development and metastasis. Therefore we explored the interaction between endothelial cells and RCC cells. Our preliminary data showed that conditioned medium (CM) from HUVEC cells activated oncogenic signaling pathways in 786-O cells, and this activity was further increased with CM from sunitinib-pretreated HUVEC cells. These data suggest the development of sunitinib resistance could be promoted by a microenviromental feedback loop. In summary, our investigations into the molecular mechanisms of sunitinib resistance demonstrated that AXL and MET were drivers of sunitinib resistance in our cell model system. We found cabozantinib inhibited both AXL and MET signaling, and suppressed cell migration. Endothelial cells also contributed to the upregulation of oncogenic signaling pathways within the tumor epithelial cell through a positive feedback loop. Ongoing experiments will further characterize the detailed mechanisms of sunitinib resistance and explore the potential utility of using MET/AXL inhibitors to treat RCC that is resistant to therapies targeting the VEGF signaling pathway. Citation Format: Lijun Zhou, Xiande Liu, Mianen Sun, Nizar M. Tannir, Douglas Laird, Eric Jonasch. The mechanisms of anti-VEGF therapy resistance in renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 687. doi:10.1158/1538-7445.AM2014-687

Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function

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.

Sunitinib to upregulate IFNg-STAT1 signaling and to increase indoleamine 2,3-dioxygenase (IDO) expression in renal cell carcinoma (RCC).

614 Background: Recently we reported that antiangiogenic therapy-treated primary RCC demonstrated increased infiltration of CD4 and CD8 T cells and higher expression of the immune checkpoint ligand PD-L1, all of which were associated with therapy resistance. IFNg-STAT1 signaling pathway drives the expression of both immunosuppressive and immunostimulatory molecules that are involved in lymphocyte trafficking, antigen presentation and immune checkpoints. The purpose of this study was to assess the impact of antiangiogenic therapy on IFNg signaling and on a broader tumor specific immune profile. Methods: We performed immunohistochemical staining of tissue microarrays (TMAs) generated from 40 sunitinib treated primary RCC from patients treated on clinical trial and compared stains to untreated control tumors. We used Vectra imaging to spectrally unmix antibody signal and to quantitate expression levels. We used immunoblot and real-time PCR to investigate the changes in protein and mRNA, respectively. We asse...

Association of resistance to antiangiogenic therapy with an immunosuppressive tumor microenvironment in metastatic renal cell carcinoma.

419 Background: Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer, and anti-angiogenic therapy is the current mainstay of treatment. RCC patients develop innate or adaptive resistance to anti-angiogenic therapy. There is a need to identify biomarkers which predict therapeutic resistance and guide combination therapy. Methods: Tissue microarrays with triplicate cores for each case were generated from 33 unaffected kidneys, 41 untreated primary RCC, and 42 bevacizumab and 39 sunitinib pretreated primary RCC from patients with metastatic RCC. Immunohistochemistry was used to visualize immune cell infiltration. Staining quantitation was performed using a Vectra multispectral system. Statistical analysis was performed using unpaired Student´s t-test. Results: We assessed the interaction between anti-angiogenic therapy and tumor immune microenvironment, and determined their impact on clinical outcome. Here we found that anti-angiogenic therapy treated RCC primary tumors demonstrated increase...

Abstract 312: Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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. Here we reported that the hypoxia inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, was in part constitutively degraded by autophagy. HIF2α interacts with autophagy protein LC3, autophagy receptor p62, and lysosome protein LAMP1. Inhibition of autophagy results in HIF2α accumulation, while induction of autophagy promotes HIF2α degradation. The E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) is required for autophagic degradation of HIF2α. There is a complementary interaction between the proteasome and autophagy in HIF2α degradation. Autophagy inactivation in ATG5 knockout cells redirects HIF2α to proteasomal degradation, while proteasome inhibition induced autophagy and favored the HIF2α-p62 interaction. Importantly, we reported that clear cell renal cell carcinoma (ccRCC) was frequently associated with mono-allelic loss and/or mutation of autophagy related gene ATG7, and low expression level of autophagy genes correlated with ccRCC progression. This study sheds light on the anticancer role for autophagy in ccRCC tumorigenesis, and reveals constitutive autophagic degradation of HIF2α as a novel tumor suppression mechanism. Citation Format: Xian-De Liu, Jun Yao, Durga N. Tripathi, Zhiyong Ding, Yi Xu, Mianen Sun, Jiangwei Zhang, Shanshan Bai, Peter German, Anh Hoang, Lijun Zhou, Xuesong Zhang, Claudio J. Conti, Eleni Efstathiou, Tony N. Eissa, Gordon B. Mills, Cheryl L. Walker, Eric Jonasch. Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 312. doi:10.1158/1538-7445.AM2014-312