Shanshan Bai

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.

The PI3K/AKT Pathway and Renal Cell Carcinoma

The phosphatidylinositol 3 kinase (PI3K)/AKT pathway is genetically targeted in more pathway components and in more tumor types than any other growth factor signaling pathway, and thus is frequently activated as a cancer driver. More importantly, the PI3K/AKT pathway is composed of multiple bifurcating and converging kinase cascades, providing many potential targets for cancer therapy. Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer that is notoriously resistant to traditional chemotherapies or radiotherapies. The PI3K/AKT pathway is modestly mutated but highly activated in RCC, representing a promising drug target. Indeed, PI3K pathway inhibitors of the rapalog family are approved for use in RCC. Recent large-scale integrated analyses of a large number of patients have provided a molecular basis for RCC, reiterating the critical role of the PI3K/AKT pathway in this cancer. In this review, we summarize the genetic alterations of the PI3K/AKT pathway in RCC as indicated in the latest large-scale genome sequencing data, as well as treatments for RCC that target the aberrant activated PI3K/AKT pathway.

Site-specific activation of AKT protects cells from death induced by glucose deprivation

The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose-deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGF receptor or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1 and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but not AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling.

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.

Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.

Agents That Stabilize Mutated von Hippel–Lindau (VHL) Protein Results of a High-Throughput Screen to Identify Compounds That Modulate VHL Proteostasis

Von Hippel–Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.

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.

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.