Search

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS–MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS–MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRASQ61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS–MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations. Significance: These findings suggest that conventional therapy–resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS–MAPK pathway.

Cancer stem cells have an important role in tumour biology. While their identity in haematological malignancies is clearly defined, stem cell identity remains elusive in some solid tumours. Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer, but the identity or existence of ccRCC stem cells remains unknown. We aimed to discern their existence using the widely utilised side population approach in ccRCC cell lines. In all cells tested, a well-defined side population was identified, and cell-based assays suggested stem-like properties. However, limiting dilution assays revealed comparable tumour initiating abilities and tumour histology of side and non-side populations, and single cell RNA-sequencing revealed minimal differences between these populations. The results indicate that the side population approach is not sufficient for cancer stem cell discovery in ccRCC.

Background The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC). Methods PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR, western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice. Results Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation. Conclusions PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.

The programmed cell death-1 (PD-1) and its ligands PD-L1 and PD-L2 are immune checkpoints. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their anti-cancer activity. Recently, PD-1 expression has been found in cancer cells. We analysed expression and functions of PD-1 in thyroid cancer (TC). Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 overexpression/stimulation promoted TC cell proliferation and migration in culture. PD-1 recruited the SHP2 phosphatase, potentiated its phosphatase activity thus enhancing Ras activation by dephosphorylation of inhibitory tyrosine 32 and triggering the MAPK cascade. PD-1 inhibition decreased, while PD-1 overexpression facilitated, TC cell xenograft growth by affecting cell proliferation. PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the Ras/MAPK pathway.

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.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide–negative counterparts, which protects ccRCC cells against natural killer cell–mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. Implications: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

Tyrosine phosphorylation of several small GTPase proteins including Ras has been reported; however, investigations to understand the biologic role of tyrosine phosphorylated small GTPases have been limited. We will present the detailed characterization of the impact of tyrosyl phosphorylation of KRas on each stage of the GTPase cycle, and on the binding to the RBD of BRaf. Using NMR and MS analysis we demonstrate that a catalytic amount of Src (1 mole of Src into 250 moles of KRas) specifically phosphorylates KRas at two of the eight tyrosine residues (Y32 and Y64) in vitro. In HEK293 cells, ectopic expression of Src with KRas results in tyrosyl phosphorylation of the same sites in KRas. Tyrosyl phosphorylation of KRas perturbs the chemical shifts of residues in the two switch regions. Using our real-time NMR-based small GTPase assay, we illustrate that tyrosyl phosphorylation alters regulation of the KRas GTPase cycle by guanine exchange factor (SOScat) and GTPase activating protein (p120GAP), rendering KRas resistant to both activities. Furthermore, using an Octet BLI binding assay, we demonstrated that tyrosyl phosphorylation of KRas strongly impairs BRaf-RBD binding. In vitro, Src-phosphorylated KRas can be completely dephosphorylated by the phosphatase SHP2. In cells, we were able to detect endogenous tyrosyl phosphorylated Ras upon SHP2 CRISPR knockout. Together, our findings demonstrate that the GTPase cycle and effector binding affinity of KRas can be regulated by the balance of Src and SHP2 activities. Citation Format: Teklab Gebregiworgis, Christopher B. Marshall, Yoshihito Kano, Michael Ohh, Mitsuhiko Ikura. Biophysical and biochemical characterization of Src-phosphorylated KRas [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A04.

In clear-cell renal cell carcinoma (ccRCC), loss of von Hippel-Lindau (VHL) tumour suppressor gene and reduced oxygen tension promote stabilisation of hypoxia-inducible factor (HIF) family of transcription factors, which promote changes in the expression of genes that contribute to oncogenesis. Multiple studies have demonstrated significant perturbations in DNA methylation in ccRCC via largely unclear mechanisms that modify the transcriptional output of tumour cells. Here, we show that the methylation status of the CpG dinucleotide within the consensus hypoxia-responsive element (HRE) markedly influences the binding of HIF and that the loss of VHL results in significant alterations in the DNA methylome. Surprisingly, hypoxia, which likewise promotes HIF stabilisation and activation, has relatively few effects on global DNA methylation. Gene expression analysis of ccRCC patient samples highlighted expression of a group of genes whose transcription correlated with methylation changes, including hypoxic responsive genes such as VEGF and TGF. These results suggest that the loss of VHL alters DNA methylation profile across the genome, commonly associated with and contributing to ccRCC progression.

There is heightened interest to devise therapies that target the oncometabolome. We show that kinase inhibitors (KIs) and biguanides synergistically target melanoma, leukemia, and breast, colon and renal cancer cells, but not non-transformed cells. Metabolic profiling confirmed opposing effects of KIs and biguanides on glycolysis, but this was insufficient to explain the observed synergy between the drugs. Rather, we define a critical role for the synthesis of non-essential amino acids (NEAA) aspartate, asparagine and serine as well as reductive glutamine metabolism, in determining the sensitivity of cancer cells to KI - biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine and serine synthesis by modulating translation of mRNAs encoding PC, ASNS, PHGDH and PSAT1. Ablation of 4E-BP1 and 2 results in a dramatic increase in serine, aspartate and asparagine levels and a substantial decrease in sensitivity of breast cancer and melanoma cells to KI - biguanide combinations. In turn, efficacy of KI - biguanide combinations is impeded by HIF1α and sustained reductive glutamine metabolism. These findings identify hitherto unappreciated translational reprograming of NEAA synthesis and HIF1α-dependent stimulation of reductive glutamine metabolism as critical metabolic vulnerabilities of cancer that underpin synergy between KIs and biguanides.

There is heightened interest to devise therapies that target the oncometabolome. We show that kinase inhibitors (KIs) and biguanides synergistically target melanoma, leukemia, and breast, colon and renal cancer cells, but not non-transformed cells. Metabolic profiling confirmed opposing effects of KIs and biguanides on glycolysis, but this was insufficient to explain the observed synergy between the drugs. Rather, we define a critical role for the synthesis of non-essential amino acids (NEAA) aspartate, asparagine and serine as well as reductive glutamine metabolism, in determining the sensitivity of cancer cells to KI - biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine and serine synthesis by modulating translation of mRNAs encoding PC, ASNS, PHGDH and PSAT1. Ablation of 4E-BP1 and 2 results in a dramatic increase in serine, aspartate and asparagine levels and a substantial decrease in sensitivity of breast cancer and melanoma cells to KI - biguanide combinations. In turn, efficacy of KI – biguanide combinations is impeded by HIF1α and sustained reductive glutamine metabolism. These findings identify hitherto unappreciated translational reprograming of NEAA synthesis and HIF1α-dependent stimulation of reductive glutamine metabolism as critical metabolic vulnerabilities of cancer that underpin synergy between KIs and biguanides.

Introduction: Neuroblastoma (NB), the most common pediatric extra-cranial solid tumor, recurs in >50% of patients who present with metastases. Strategies to treat relapsed NB, which is often fatal, include targeting relapse-specific signaling pathways. Recently, sequencing studies have revealed that 78% of mutations detected in relapse samples are predicted to activate the RAS-MAPK pathway, suggesting there may be opportunities to pharmacologically target this pathway to treat recurrent NB. Experimental Design: Our lab previously reported that the PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and pharmacologic inhibition of SHP2 inhibited glioblastoma growth in vivo. To determine whether inhibiting SHP2 in NB would impact tumor growth we treated a panel of NB cells with diverse genetic backgrounds, including mutations in NRAS, with different SHP2 inhibitors (SHP099 and II-B08) alone and in combination with additional drugs that target RAS-MAPK signaling. Results: In comparison to NB cell lines harboring endogenous NRAS mutations (NRASmt), cells with NRAS wild-type (NRASwt) were more sensitive to SHP2 inhibitors. In addition, NRASwt cells engineered to overexpress NRASmt were more resistant to SHP099 than cells with endogenous or overexpressed NRASwt, as demonstrated by higher levels of proliferation, increased survival, and diminished apoptosis. Furthermore, SHP099 effectively inhibited SHP2 in both NRASwt and NRASmt cells, but only failed to inactivate the downstream RAS effector ERK1/2 in NRASmt cells, suggesting that SHP099 treatment alone is ineffective in cells harboring NRAS mutations. To determine whether SHP099 resistance in NRASmt cells could be overcome with combination strategies, sensitive and resistant NB cells were treated with SHP2 inhibitors alone or in combination with other RAS-MAPK pathway inhibitors (vemurafenib, trametinib and ulixertinib). Interestingly, in comparison to SHP099 or II-B08 alone, all three combinations significantly reduced survival and IC50 values in both NRASwt and NRASmt cells. Using the Bliss independence model we determined all three combinations were synergistic. Conclusions: Our studies demonstrate that NRAS mutations are associated with resistance to SHP2 inhibitors, and that in certain tumors, based on the genetic status of RAS-MAPK-SHP2 signaling components, combinations of drugs targeting this pathway could be effective strategies for relapsed NB. Citation Format: Ivette Valencia-Sama, Teresa Adderley, Lynn Kee, Gabriella Christopher, Yoshihito Kano, Michael Ohh, Meredith Irwin. Targeting SHP2 and RAS MAPK pathway in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3123.

Loss of von Hippel–Lindau protein (pVHL) is known to contribute to the initiation and progression of tumours associated with VHL disease as well as certain sporadic tumours including clear cell renal cell carcinoma (ccRCC). The VHL gene was first identified and cloned over 20years ago and our understanding of its functions and effects has significantly increased since then. The best-known function of pVHL is its role in promoting the degradation of hypoxia-inducible factor α subunit (HIFα) as part of an E3 ubiquitin ligase complex. HIF stabilisation and transcriptional activation are also associated with various epigenetic alterations, indicating a potential role for VHL loss with changes in the epigenome. This review will highlight current knowledge regarding pVHL as well as discuss potentially novel roles of pVHL and how these may impact on cancer progression.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm in children characterized by the overproduction of monocytic cells that infiltrate the spleen, lung, and liver. JMML remains a disease for which few curative therapies are available other than myeloablative hematopoietic stem cell transplant (HSCT); however, relapse remains a major cause of treatment failure and the long-term morbidities of HSCT for survivors are substantial. A hallmark feature of JMML is acquired hypersensitivity by clonal myeloid progenitor cells to granulocyte macrophage-colony stimulating factor (GM-CSF) via a largely unknown mechanism. Here, we identify c-Cbl (henceforth referred to as Cbl) as a GM-CSF receptor (GMR) adaptor protein that targets Src for ubiquitin-mediated destruction upon GM-CSF stimulation and show that a loss of negative regulation of Src is pivotal in the hyperactivation of GMR signaling in Cbl-mutated JMML cells. Notably, dasatinib, an U.S. Food and Drug Administration–approved multikinase inhibitor that also targets Src family, dramatically attenuated the spontaneous and GM-CSF-induced hypersensitive growth phenotype of mononuclear cells from peripheral blood and bone marrow collected from JMML patients harboring Cbl or other known JMML-associated mutations. These findings reveal Src kinase as a critical oncogenic driver underlying JMML. Cancer Res; 73(8); 2540–50. ©2013 AACR.

DNA repair is essential for maintaining genomic stability, and defects in this process significantly increase the risk of cancer. Clear-cell renal cell carcinoma (CCRCC) caused by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is characterized by high genomic instability. However, the molecular mechanism underlying the association between the loss of VHL and genomic instability remains unclear. Here, we show that suppressor of cytokine signaling 1 (SOCS1) promotes nuclear redistribution and K63-ubiquitylation of VHL in response to DNA double-strand breaks (DSBs). Loss of VHL or VHL mutations that compromise its K63-ubiquitylation attenuates the DNA-damage response (DDR), resulting in decreased homologous recombination repair and persistence of DSBs. These results identify VHL as a component of the DDR network, inactivation of which contributes to the genomic instability associated with CCRCC.

There are 17 known ubiquitin-like proteins (UBLs) from nine phylogenetically distinct classes (NEDD8, SUMO, ISG15, FUB1, FAT10, Atg8, Atg12, Urm1, and UFM1) that have been identified to conjugate to substrates in a manner analogous to ubiquitin. NEDD8 is one of the most studied UBLs and shares the highest amino acid similarity to ubiquitin. Here, we review the current knowledge of the NEDD8 conjugation cascade derived from functional studies in genetic model organisms, structural insights from crystallographic studies, biochemical studies identifying a growing list of NEDD8 substrates with oncogenic implications, and attempts to pharmacologically target the NEDD8 pathway in cancer.

Summary There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.

ΔNp63α is a critical pro-survival protein overexpressed in 80% of head and neck squamous cell carcinomas (HNSCCs) where it inhibits TAp73β transcription of p53-family target genes, which is thought to increase HNSCC resistance to chemotherapy-induced cell death. However, the mechanisms governing ΔNp63α function are largely unknown. In this study, we identify special AT-rich-binding protein 2 (SATB2) as a new ΔNp63α-binding protein that is preferentially expressed in advanced-stage primary HNSCC and show that SATB2 promotes chemoresistance by enhancing ΔNp63α-mediated transrepression by augmenting ΔNp63α engagement to p53-family responsive elements. Furthermore, SATB2 expression positively correlates with HNSCC chemoresistance, and RNA interference-mediated knockdown of endogenous SATB2 re-sensitizes HNSCC cells to chemotherapy- and γ-irradiation-induced apoptosis, irrespective of p53 status. These findings unveil SATB2 as a pivotal modulator of ΔNp63α that governs HNSCC cell survival.

More than 30% of all human cancers have Ras mutations and more than 95% of pancreatic cancers harbor KRAS mutations. However, therapeutically targeting cancers driven by oncogenic Ras mutations is still an ongoing investigation. KRas is a small GTPase found in either a GDP-bound inactive form or a GTP-loaded activated form. The activated form of KRas localizes on the membrane where it binds and activates downstream effector proteins such as Raf kinases. GDP-bound Ras can be activated by nucleotide exchange of GTP for GDP, a reaction that is catalyzed by guanine nucleotide exchange factors (GEFs). Ras is inactivated by hydrolysis of GTP, which is assisted by GTPase activating proteins (GAPs). It was recently reported that Ras can be tyrosyl phosphorylated by Src and dephosphorylated by SHP2 (PNAS 2014;111(36):E3785-94; Nat Commun 2015;6:8859). Here we have characterized the structural and functional alterations of Src-phosphorylated KRas to mechanistically explain the impact of tyrosyl phosphorylation on the GTPase cycle. Our NMR and MS analyses show that Src phosphorylates KRas at Tyr32 and Tyr64, which perturbs the chemical shifts of several residues in each of the two "switch" regions that mediate interactions with effectors and regulators. Using real-time NMR and biolayer interferometry (Octet) assays, we demonstrated the negative impact of KRas phosphorylation on the GTPase cycle as well as BRAF binding. Conversely, the SH2 domain containing inositol 5-phosphatase 2 (SHP2) dephosphorylates KRAS and reverses Src-induced phosphorylation. In vivo, either the pharmacologic inhibition or genetic ablation of SHP2 promotes cell death in several KRAS mutant cancers. Our findings reveal that altering the KRas GTPase cycle regulation via the balance of Src and SHIP2 activities may create a therapeutic vulnerability for pancreatic cancer. Citation Format: Teklab Gebregiworgis, Christopher B. Marshall, Yoshihito Kano, Nikolina Radulovich, Ming-Sound Tsao, Michael Ohh, Mitsuhiko Ikura. Altering the regulation of KRAS GTPase cycle via Src and SHP2 creates a potential therapeutic vulnerability for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4360.

PURPOSE OF REVIEW To provide an overview of the recent advances in the understanding of the molecular mechanisms governing the tumour suppressor functions of the von Hippel-Lindau protein. RECENT FINDINGS von Hippel-Lindau is a vital component of an E3 ubiquitin ligase complex involved in the oxygen-dependent targeting of hypoxia-inducible factor for ubiquitin-mediated destruction. Recent reports have linked von Hippel-Lindau to the regulation of diverse biological processes including cell adhesion, extracellular matrix assembly and ciliogenesis in a manner dependent and/or independent of hypoxia-inducible factor. SUMMARY The tumour suppressor function of von Hippel-Lindau has remained hypoxia-inducible factor-centric since the discovery of von Hippel-Lindau as a bona fide negative regulator of the ubiquitous oxygen-sensing pathway. Emerging evidence supports this hypothesis with the elucidation of fundamental cellular processes deregulated upon the inactivation of the von Hippel-Lindau-hypoxia-inducible factor pathway, but has also proved compelling on the hypoxia-inducible factor-independent tumour suppressor role of von Hippel-Lindau. These and continuing studies into the molecular pathways and mechanisms governing the tumour suppressor functions of von Hippel-Lindau will ultimately afford new avenues for anticancer strategies for the improved treatment of a diverse array of cancers.

The most prevalent mutations associated with the development of clear-cell renal cell carcinoma (CC-RCC) are the loss-of-function mutations of von Hippel-Lindau (VHL) tumor suppressor gene. These mutations invariably result in an inappropriate accumulation of HIF-alpha due to a failure of VHL as a substrate-recognition component of an E3 ubiquitin ligase complex to target HIFalpha for oxygen-dependent ubiquitin-mediated destruction. Stabilization of HIF-2alpha, but not HIF-1alpha, is the critical oncogenic event upon the functional loss of VHL in the development of CC-RCC. Here, we show that HIF-3alpha4, an alternatively spliced variant of human HIF-3alpha with similar domain structure as the murine inhibitory PAS protein (IPAS), forms an abortive transcriptional complex with HIF-2alpha and prevents the engagement of HIF-2 to the hypoxia-responsive elements (HREs) located in the promoter/ enhancer regions of hypoxia-inducible genes. In addition, the re-expression of HIF-3alpha4 in VHL-null 786-O CC-RCC cells via adenovirus decreases the endogenous expression of HIF-2-driven gene expression and suppresses the growth of 786-O tumor xenografts in SCID mice. These results suggest that HIF-3alpha4 is a naturally occurring dominant-negative HIF-3alpha splice isoform with tumor suppressive activity and support the targeted delivery of HIF-3alpha4 as a potential therapeutic option to curtail HIF-dependent tumor progression.

Loss of the von Hippel-Lindau (VHL) tumor suppressor gene contributes to proliferative disorders including renal cell carcinoma. The consequence of VHL loss is increased levels of hypoxia-inducible factor-alpha (HIFalpha), which is targeted for proteolytic degradation by the VHL gene product pVHL. HIF is a transcription factor that increases the expression of factors critical for tumorigenesis in renal cell carcinoma. We report here another regulatory component of HIFalpha expression in renal cancer cells. Phospholipase D (PLD), which is commonly elevated in renal and other cancers, is required for elevated levels of both HIF1alpha and HIF2alpha in VHL-deficient renal cancer cells. The induction of both HIF1alpha and HIF2alpha by hypoxic mimetic conditions was also dependent on PLD in renal cancer cells with restored pVHL expression. The effect of PLD activity upon HIFalpha expression was at the level of translation. PLD activity also provides a survival signal that suppresses apoptosis induced by serum deprivation in the renal cancer cells. Suppression of HIF2alpha has been shown to reverse tumorigenesis with renal cancer cells. The finding here that HIF2alpha expression is dependent on PLD in renal cancer cells suggests that targeting PLD signals may represent an alternative therapeutic strategy for targeting HIF2alpha in renal cancers where HIF2alpha is critical for tumorigenesis and elevated PLD activity is common.

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras., Aberrant Ras signalling resulting in downstream Mek/Erk pathway activation is found in many cancers. Here, the authors show that the phosphatase SHP2 dephosphorylates Ras resulting in increased Ras activity, and that increased SHP2 activity is found in glioblastomas.

ECV is an E3 ubiquitin ligase complex, which is composed of elongins B and C, Rbxi, Cul2, the substrate-conferring von Hippel-Lindau (VHL) tumorsuppressor protein that targets the catalytic α subunit of hypoxia-inducible factor (HI F) for oxygen-dependent ubiquitin-mediated destruction. Mutations in VHL that compromise proper HIFα regulation through ECV have been documented in the majority of renal cell carcinomas, underscoring the significance of the VHL-HIF pathway in renal epithelial oncogenesis. Recent evidence has shown that the modification of Cul2 by the ubiquitin-like molecule NEDD8 increases the activity of ECV to ubiquitylate HIFα. However, the underlying mechanism responsible for the NEDD8-mediated induction of ECV function is unknown. Here, we demonstrate that oxygen-dependent recognition of HIFα by VHL triggers Rbxi-dependent neddylation of Cul2, which preferentially engages the E2 ubiquitin-conjugating enzyme UbcH5a. These events establish a central role for the neddylation of Cul2 in a previously unrecognized, temporally coordinated activation of ECV with the recruitment of its substrate HIFα.

Hypoxia-inducible factor (HIF) is a central regulator of cellular responses to hypoxia, and under normal oxygen tension the catalytic α subunit of HIF is targeted for ubiquitin-mediated destruction via the VHL-containing E3 ubiquitin ligase complex. Principally known for its association with oncogenesis, HIF has been documented to have a role in the antibacterial response. Interferons, cytokines with antiviral functions, have been shown to upregulate the expression of HIF-1α, but the significance of HIF in the antiviral response has not been established. Here, using renal carcinoma cells devoid of VHL or reconstituted with functional wild-type VHL or VHL mutants with various abilities to negatively regulate HIF as an ideal model system of HIF activity, we show that elevated HIF activity confers dramatically enhanced resistance to vesicular stomatitis virus (VSV)-mediated cytotoxicity. Inhibition of HIF activity using a small-molecule inhibitor, chetomin, enhanced cellular sensitivity to VSV, while treatment with hypoxia mimetic CoCl2promoted resistance. Similarly, targeting HIF-2α by RNA interference also enhanced susceptibility to VSV. Expression profiling studies show that upon VSV infection, the induction of genes with known antiviral activity, such as that encoding beta interferon (IFN-β), is significantly enhanced by HIF. These results reveal a previously unrecognized role of HIF in the antiviral response by promoting the expression of the IFN-β gene and other genes with antiviral activity upon viral infection.

The physiologic response to changes in cellular oxygen tension is ultimately governed by a heterodimeric transcription factor called hypoxia-inducible factor (HIF), which, in adaptation to compromised oxygen availability, transactivates a myriad of genes, including those responsible for de novo vascularization, production of oxygen-carrying red blood cells, and anaerobic metabolism. Accumulation of HIF is observed in most types of solid tumors and is frequently associated with poor prognosis and disease progression, underscoring the importance and relevance of HIF in cancer. The protein stability and, thereby, the activity of HIF are principally regulated by the von Hippel-Lindau (VHL) tumorsuppressor-containing E3 ubiquitin ligase complex (ECV) that targets the catalytic subunit HIFα for oxygendependent ubiquitin-mediated destruction. Individuals who inherit germline VHL mutation develop VHL disease, which is characterized by the development of hypervascular tumors in multiple yet specific organs. This review will examine recent progress in our understanding of the molecular mechanisms governing the function of ECV and the significance of consequential regulation of HIF in oncogenesis.

The von Hippel-Lindau (VHL) tumour suppressor gene encodes a substrate-specifying component of an E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) alpha subunits for degradation under normoxia. The VHL protein is composed of an N-terminal HIFalpha-binding beta domain and a C-terminal alpha domain, which is necessary and sufficient for the formation of the E3 multiprotein enzyme. A large number of disease-causing mutations in either the alpha or beta domain renders HIFalpha stable irrespective of oxygen tension, leading to the upregulation of numerous HIF-target genes, such as GLUT1 and VEGF. Here, we show that VHL forms a self-associated complex in vivo, but not in vitro, and demonstrate that coexpression of two different VHL missense mutants -- one in the alpha domain and the other in the beta domain -- restores HIF-mediated gene expression profile. These findings indicate that VHL homotypic complexes can function in vivo in a complementary fashion to target HIFalpha for ubiquitin-mediated proteolysis, and potentially explain why VHL-associated tumours with a missense mutation-carrying VHL allele is almost invariably accompanied by a second VHL allele harbouring a gross truncation or deletion.

The development of hereditary von Hippel-Lindau (VHL) disease and the majority of sporadic kidney cancers are due to the functional inactivation of the VHL gene. The product of the VHL gene, pVHL, in association with elongins B and C, cullin 2, and Rbx1 form an E3 ubiquitin-ligase complex VEC that targets the alpha subunits of hypoxia-inducible factor (HIF) for ubiquitination. Ubiquitin-tagged HIF-α proteins are subsequently degraded by the common 26S proteasome. pVHL functions as the substrate-docking interface that specifically recognizes prolyl-hydroxylated HIF-α. This hydroxylation occurs only in the presence of oxygen or normoxia. Thus, under hypoxia, HIF-α subunits are no longer subjected to degradation and are thereby able to dimerize with the common and constitutively stable β subunits. The heterodimeric HIFs upregulate a myriad of hypoxia-inducible genes, triggering our physiologic response to hypoxia. Inappropriate accumulations of HIF-α in VHL disease are believed to contribute to the pathogenesis via the upregulation of several of these HIF target genes. Our current molecular understanding of the roles of HIF and pVHL in the development of VHL-associated clear-cell renal cell carcinoma (CC-RCC) is the focus of this review.

von Hippel-Lindau (VHL) tumor suppressor protein—inactivated in VHL disease and sporadic kidney cancer—is a component of an E3 ubiquitin ligase complex that selectively ubiquitinates the α subunit of the hypoxia-inducible factor (HIF) transcription factor for subsequent destruction by the 26S proteasome. Here, we report the identification and characterization of the first VHL homologue, VHL-like protein (VLP), located on chromosome 1q21.2. A 676-bp partial cDNA encoding a 139-amino acid protein that is 78% similar to VHL was isolated by reverse transcription-PCR from human brain cerebellum and several cancer cell lines. The expression of VLP transcript is most abundant in the placenta. Like VHL, VLP contains a β domain capable of binding HIFα. However, unlike VHL, it does not contain a recognizable α domain, which is required for nucleating the multiprotein E3 ubiquitin ligase complex. The increased expression of VLP in the presence of VHL attenuated the ubiquitination of HIFα and led to the accumulation of downstream HIF target genes. These results taken together indicate that VLP functions as a dominant-negative VHL to serve as a protector of HIFα.

Protein synthesis is one of the most energy consuming process in the cell. Oncogenic kinases (e.g. EGFR/HER2, BCR/ABL and BRAF) play a central role in reprogramming translation and energy metabolism in neoplasia, whereby cancer cells must provide sufficient ATP to support increased levels of protein synthesis required for neoplastic growth. The downstream mechanisms that link translational machinery and energy homeostasis in cancer, however, remain largely unknown. We found that widely used anti-diabetics (biguanides) abrogate adaptations to EGFR/HER2 inhibitor-induced energetic stress, which results in synergistic anti-neoplastic effects both in vitro and in vivo. In turn, breast cancer cells in which 4E-BP1/2 expression was abrogated by CRISPR were partially resistant to the combination of EGFR/HER2 inhibitors and biguanides. This was paralleled by the inability of the drugs to inhibit the eIF4F complex assembly and translation of mRNAs encoding important metabolic regulators including those involved in serine biogenesis (PHGDH, PSAT1) and one carbon metabolism (MTHFD1L). Comparable results were observed when BRAF and BCR/ABL inhibitors were combined with biguanides, which suggests that translational regulation of metabolic genes via the mTORC1/4E-BE/eIF4E pathway plays a major role in energy stress response in cancer. Together our findings demonstrate that the eIF4F complex is an important mediator of metabolic adaptation in response to the combination of biguanides and clinically-used kinase inhibitors and suggest that the efficiency of such anti-cancer strategies are dependent on the integrity of the translation initiation machinery. Citation Format: Laura Hulea, Marie Cargnello, Simon-Pierre Gravel, Young Im, Shannon McLaughlan, Yunhua Zhao, Jenna Ching, Yutian Cai, Ola Larsson, Michael Ohh, Josie Ursini-Siegel, Julie St-Pierre, Michael Pollak, Ivan Topisirovic. eIF4F links translation to energy stress response in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A31.

E2-EPF ubiquitin carrier protein (UCP) is a member of an E2 family of enzymes that catalyzes the ligation of ubiquitin to proteins targeted for destruction by the proteasome. UCP is overexpressed in common human cancers, suggesting its involvement in oncogenesis, but a physiologic target of UCP has not been identified. In a recent report published in Nature Medicine, Jung et al. identified von Hippel-Lindau (VHL) tumor suppressor protein, which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction, as a bona fide substrate of UCP and demonstrated a potential pVHL-HIF pathway-dependent role for UCP in cancer development.

Background and Aims: Mutations in RAS and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers, including up to 25% of lung cancers and over 90% of pancreatic cancers. Ras function as ‘molecular switches’ in a number of signaling pathways that regulate vital cellular functions. Over the past few decades, it has become clear that the activity or the oncogenic potential of Ras is dependent on the non-receptor tyrosine kinase Src to regulate essential cellular pathways for proliferation, differentiation and survival of eukaryotic cells. Recently, we showed that Src binds to and phosphorylates Ras on a conserved tyrosine residue at position 32 within the switch I region to promote Ras GTPase activity. We then identified Shp2 as the critical tyrosine phosphatase that is responsible for the dephosphorylation and activation of Ras. However, it is unknown whether pharmacologic manipulation of Shp2 activity would suppress the growth of Ras-driven pancreatic cancer (PDACs), a highly aggressive disease currently without effective treatment options or cure. Methods: We generated the available Ras, Src, and SHP2 reagents (wild-type, oncogenic, truncation, dominant-negative, catalytically active or inactive, mutants) to determine the structure/function relationship between Ras, Src, and SHP2. The interaction was assessed by immunoprecipitation and western blotting. We utilized several PDAC cell lines and patient-derived xenografts cell lines (PDX-C). The cell growth was evaluated by Alarmar Blue assay. Results: We identified SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signaling. In comparison to K-Ras WT pancreatic cancer cells, the level of phospho-Shp2 was observed to be higher in K-Ras mutated cell lines in both absence and presence of growth factors. Pharmacologic inhibition of Shp2 using a specific cell-permeable Shp2 inhibitor decreased the level of phosphorylation of Shp2 as well as downstream signaling such as pAKT and pERK especially in the presence of growth factor. Notably, we show that treatment with this inhibitor suppressed the cell proliferation in both K-Ras WT and mutated pancreatic cancer cells as well as PDX-C in a caspase-dependent manner. Conclusion: Our results identify SHP2 as a direct activator of Ras and potential therapeutic target for pancreatic cancer driven by a previously ‘undruggable’ oncogenic or hyperactive Ras. Citation Format: Yoshihito Kano, Severa Bunda, Jen Jen Yeh, Zhong-Yin Zhang, Michael Ohh.{Authors}. Novel treatment strategy for pancreatic cancer by targeting the ‘undruggable’ Ras oncoprotein. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B13.

A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property - known as the "Warburg effect" or aerobic glycolysis - contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxia-inducible factor (HIF) - a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the α subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.

Tumour hypoxia promotes the accumulation of the otherwise oxygen-labile hypoxia-inducible factor (HIF)-alpha subunit whose expression is associated with cancer progression, poor prognosis and resistance to conventional radiation and chemotherapy. The oxygen-dependent degradation of HIF-alpha is carried out by the von Hippel-Lindau (VHL) protein-containing E3 that directly binds and ubiquitylates HIF-alpha for subsequent proteasomal destruction. Thus, the cellular proteins involved in the VHL-HIF pathway have been recognized as attractive molecular targets for cancer therapy. However, the various compounds designed to inhibit HIF-alpha or HIF-downstream targets, although promising, have shown limited success in the clinic. In the present study, we describe the bioengineering of VHL protein that removes the oxygen constraint in the recognition of HIF-alpha while preserving its E3 enzymatic activity. Using speckle variance-optical coherence tomography (sv-OCT), we demonstrate the dramatic inhibition of angiogenesis and growth regression of human renal cell carcinoma xenografts upon adenovirus-mediated delivery of the bioengineered VHL protein in a dorsal skin-fold window chamber model. These findings introduce the concept and feasibility of 'bio-tailored' enzymes in the treatment of HIF-overexpressing tumours.

p53 mutations are rarely detected in clear cell renal cell carcinoma (CCRCC), but, paradoxically, these tumors remain highly resistant to chemotherapy and death receptor–induced death. Here, we show that the accumulation of hypoxia-inducible factor 2α (HIF2α), a critical oncogenic event in CCRCC following the loss of von Hippel-Lindau (VHL) tumor suppressor protein, leads to Hdm2-mediated suppression of p53. Primary CCRCC specimens exhibiting strong hypoxic signatures show increased levels of activated nuclear phospho-Hdm2(Ser166), which is concomitant with low p53 expression. The abrogation of Hdm2-p53 interaction using the small-molecule Hdm2 inhibitor nutlin-3 or the downregulation of HIF2α via HIF2α-specific short hairpin RNA or wild-type VHL reconstitution restores p53 function and reverses the resistance of CCRCC cells to Fas-mediated and chemotherapy-induced cell death. These findings unveil a mechanistic link between HIF2α and p53 and provide a rationale for combining Hdm2 antagonists with chemotherapy for the treatment of CCRCC. [Cancer Res 2009;69(23):9056–64]

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma., Author Summary Together, renal oncocytoma and chromophobe renal cell carcinoma (RCC) account for approximately 10% of masses that are resected from the kidney. However, the molecular defects that are associated with the development of these neoplasias are not clear. Here, we take advantage of recent advances in genetics and computational analysis to screen for chromosomal abnormalities that are present in both renal oncocytoma and chromophobe RCC. We show that while chromophobe RCC cells contain an extra copy of chromosome 19, the renal oncoctyoma cells contain a rarely reported chromosomal abnormality. Both of these chromosomal abnormalities result in transcriptional disruptions of EGLN2, a gene that is located on chromosome 19 and is critical for the cellular response to changes in oxygen levels. Defects in oxygen sensing are found in other types of kidney tumors, and the identification of EGLN2 directly implicates defects in the oxygen-sensing network in these neoplasias as well. These findings are important because the chromosomal defect present in renal oncocytomas may also be present in other tumor cells. In addition, deregulation of EGLN2 reveals a unique way in which perturbations in oxygen-sensing are associated with disease.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene results in highly vascularized tumors, making the VHL tumor syndrome an ideal system to study the mechanisms of angiogenesis. VHL operates along two pathways with the first involving hypoxia-inducible factor-α degradation and down-regulation of its proangiogenic target genes vascular endothelial growth factor and platelet-derived growth factor-β, and the second pathway promoting extracellular matrix (ECM) assembly. Secretion of proangiogenic factors was shown to be a primary inducer of angiogenesis. Here, we show that loss of ECM assembly correlates with tumor angiogenesis in VHL disease. Upon inactivation of the VHL-ECM assembly pathway, we observe tumors that are highly vascularized, have a disrupted ECM, and show increased matrix metalloproteinase-2 activity. Loss of the VHL pathway leading to hypoxia-inducible factor-α degradation results in tumors with increased vascular endothelial growth factor levels but with surprisingly low microvessel density, a tightly assembled ECM and low invasive ability. We conclude that loss of ECM integrity could promote and maintain tumor angiogenesis by providing a route for blood vessels to infiltrate tumors. (Cancer Res 2006; 66(3): 1313-9)

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2α did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF. (Cancer Res 2006; 66(3): 1553-60)

Renal clear-cell carcinoma (RCC) is the predominant form of kidney cancer and is highly refractory to conventional anti-cancer therapies. The status of p53 tumor suppressor gene has been correlated with the efficacy of radio- and chemotherapies, where presence of mutant p53 is associated with reduced responsiveness to treatment. However, p53 itself is rarely mutated in RCC, rather suggesting that the p53 pathway might be compromized in RCC cells. In support of this notion, the transactivation property of normal p53 was shown to be repressed in various transformed kidney epithelial cells via an unknown dominant-negative mechanism. Mutation of the von Hippel-Lindau ( VHL ) gene causes familial VHL disease, which includes predisposition to RCC. Moreover, biallelic inactivation of VHL has been observed in the vast majority of sporadic RCC. Recently, the expression of pVHL in RCC cells was demonstrated to elevate the expression of p53 by inducing the binding of RNA-stabilizing protein HuR to the 3′untranslated region of p53 mRNA. Contrary to this finding, we report here that the reconstitution of a variety of VHL (−/−) RCC lines including 786-O, RCC4, and A498 or non-RCC cells with wild-type pVHL does not influence the expression of p53 and fails to induce p53-responsive gene p21 CIP1/WAF1 or p53-responsive reporters. These results suggest that the expression of p53 in RCC cells is independent of pVHL.

The majority of kidney cancers are caused by the mutation of the von Hippel-Lindau ( VHL) tumor suppressor gene. VHL protein (pVHL) is part of an E3 ubiquitin ligase complex called VEC that is composed of elongin B, elongin C, cullin 2, NEDD8, and Rbx1. VEC targets a hypoxia-inducible factor (HIF) transcription factor for ubiquitin-mediated destruction selectively in the presence of oxygen. In the absence of wild-type pVHL, as in VHL patients or in the majority of sporadic clear cell renal cell carcinomas, HIF-responsive genes are inappropriately activated even under normoxia. Recent insights into the molecular mechanisms regulating the function of pVHL, and thereby HIF, in the context of kidney cancer are the focus of this review.

Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and have also been identified in several familial/developmental syndromes. Ras is also an important factor in many cancers where it is not mutated but functionally activated through inappropriate function of other signal transduction elements. One such example is the overexpression of growth factor receptors in glioblastoma (GBM). Elucidating the mechanistic details of the GTPase reaction has been an important scientific goal and the subject of intensive research for many years. However to this day, targeting Ras directly remains elusive. Our laboratory recently demonstrated that Src binds to and phosphorylates Ras on a conserved tyrosine residue markedly reducing the binding of effector Raf to Ras and concomitantly increasing the rate of GTP hydrolysis leading to Ras inactivation. Furthermore we demonstrate that tyrosine phosphorylated Ras can be dephosphorylated by the phosphatase SHP2 resulting in Ras activation. We also demonstrate that increased Ras activity in human primary GBM cell lines and astrocytes derived from GBM-prone mice, as well as in human GBM patient samples results from elevated SHP2 activity. Finally we show that the highly specific SHP2 inhibitor II-B08 promotes tyrosine phosphorylation of Ras leading to attenuation of downstream proliferative MAPK/ERK signaling and reduction in tumor burden in mouse models of GBM.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors observed in this disorder include retinal and central nervous system hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL gene product, pVHL, is a component of a ubiquitin ligase which targets the transcription factor known as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. pVHL also plays roles in the control of extracellular matrix formation and cell-cycle exit. Different VHL mutations confer different site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of pheochromocytoma without the other stigmata of VHL disease. Here we report that the products of such type 2C VHL alleles retain the ability to down regulate HIF but are defective for promotion of fibronectin matrix assembly. Furthermore, pVHL L188V, a well studied type 2C mutant, retained the ability to suppress renal carcinoma growth in vivo. These studies strengthen the notion that HIF deregulation plays a causal role in hemangioblastoma and renal carcinoma, and raises the possibility that abnormal fibronectin matrix assembly contributes to pheochromocytoma pathogenesis in the setting of VHL disease.

Kaelin WG, Iliopoulos O, Lonergan KM, Ohh M (Dana-Farber Cancer Institute, Brigham and Women's Hospital, Boston, MA, USA). Functions of the von Hippel–Lindau tumour suppressor protein (Minisymposium: MEN & VHL). J Intern Med 1998; 243: 535–9. Von Hippel–Lindau disease (VHL) is caused by germline mutations in the VHL tumour suppressor gene. Tumour development in this setting is due to loss or inactivation of the remaining wild-type VHL allele. The VHL gene product (pVHL) resides primarily in the cytoplasm. A frequently mutated region of pVHL can bind to complexes containing elongin B, elongin C and Cul2. Loss of pVHL leads to an inappropriate accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This finding is most likely to account for the hypervascular nature of VHL-associated neoplasms. Current studies are focussed on understanding if and how binding to elongins and Cul2 is linked to the ability of pVHL to regulate hypoxia-inducible mRNAs. In this regard, it is perhaps noteworthy that elongin C and Cul2 are homologous to yeast proteins Skp1 and Cdc53. These latter proteins participate in the formation of complexes that target certain proteins for ubiquitination.

Cell-cell adhesion is critical in the generation of effective immune responses and is dependent upon the expression of a variety of cell surface receptors. Intercellular adhesion molecule-1 (ICAM-1; CD54) is an inducible cell surface glycoprotein expressed at a low level on a subpopulation of hematopoietic cells, vascular endothelium, fibroblasts, and certain epithelial cells. However, its expression is dramatically increased at sites of inflammation, providing important means of regulating cell-cell interactions and thereby inflammatory responses. Inasmuch the modulation of ICAM-1 expression during inflammation by pharmacologic agents might be very attractive for medical treatment, the intracellular regulatory elements and signaling pathways underlying the inducible expression of ICAM-1 by proinflammatory cytokines remain largely unknown. In this review, a novel posttranscriptional regulation of ICAM-1 gene expression by two inflammatory mediators, interferon-gamma and phorbol myristate acetate, and the possible role of the serine/threonine phosphorylation pathway in the cycloheximide-induced ICAM-1 message stabilization are discussed in light of our current understanding of ICAM-1 gene regulation during an inflammatory response.

Tumor hypoxia is associated with disease progression, drug resistance and poor prognosis. It has been shown previously that hypoxia prolongs the activation of epidermal growth factor receptor (EGFR) by delaying clathrin-dependent endocytosis-mediated deactivation of receptors. Recently, caveolin (CAV) mediated signaling and trafficking have been reported to be involved in the oncogenesis of many tumours, including clear cell renal cell carcinoma (CCRCC). However, the role of CAV-1 in the tumor progression is unclear. Here, we show that the loss of von Hippel-Lindau (VHL) protein – the principal negative regulator of hypoxia-inducible factor (HIF) – increases the auto-phosphorylation of epidermal growth factor receptor (EGFR), thus attributing to an enhanced ligand-independent cell signaling pathway, majorly Ras-Raf-MEK-ERK pathway, and leading to cell proliferation. The auto-phosphorylation of EGFR is due to the HIF-dependent overexpression of caveolin-1 (CAV-1) at the level of transcription, which enhances the association between EGFR and CAV-1 and the auto-dimerization of EGFR. Primary CCRCC tumors and numerous hypoxia treated tumor cell lines exhibit significantly higher expression of CAV-1 protein. Using a dorsal skin-fold window chamber on SCID mice, shCAV-1 knockdown 786-O tumor cell line exhibits lower growth than that of shscramble counterpart in xenografts. These findings support a model in which tumour hypoxia or oncogenic activation of HIF prolongs ligand-independent RTK signaling through the over-expression of CAV-1. Given the oncogenic role of CAV-1, it should be considered as a future therapeutic target in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1973. doi:10.1158/1538-7445.AM2011-1973

Caveolin-1 (Cav-1) is a multifunctional scaffolding protein with multiple binding partners that is associated with cell surface caveolae and the regulation of lipid raft domains. Cav-1 regulates multiple cancer-associated processes including cellular transformation, tumour growth, cell death and survival, and multidrug resistance. A high expression of Cav-1 is found in various cancer , including clear cell renal cell carcinoma (CCRCC), correlating with large, high grade tumors and poor prognosis. However, the role of Cav-1 in the oncogenesis of CCRCC remains unclear, and its role as either an oncogene or tumour suppressor is still under debate. Here, we investigated the mechanism of Cav-1 in controlling cell proliferation using in vitro and in vivo assays. We report that Cav-1 is highly expressed in tumour compared to normal tissue in patients with CCRCC, thus Cav-1 likely functions as an oncogene. Upon knock-down of Cav-1, tumour cells (786-VHL-/-) showed a decrease in proliferation. We showed that Cav-1 is associating with EGFR, a receptor tyrosine kinase, to activate the Ras-RAF-MEK-ERK kinase pathway and induce cell proliferation. We found that Cav-1 binds to EGFR in immunoprecipitation. The Ras pathway is upregulated in cells overexpressing Cav-1 after EGF stimulation. Upon knockdown of Cav-1, the level of p-ERK, p-MEK, and p-c-Raf decreased, as well as the rate of cell proliferation. Upstream of the Ras pathway, we report that Cav-1 appears to be under the control of hypoxia-inducible factor (HIF). Under hypoxia, VHL (the principal negative regulator of HIF) is downregulated, thus leading to upregulation of HIF. We showed that under hypoxia, Cav-1 increased in 786-VHL, Hela and A431 cell lines. Similarly, cells with VHL loss (786-VHL-/-), thus high HIF level, had high levels of Cav-1. We are currently examining the role of Cav-1 in tumour growth by injecting SCID mice with either 786-VHL-/-, or Cav-1 knock-down 786-VHL-/- tumour cells. Since Cav-1 level seems to be under the control of HIF in renal, cervical and squamous carcinoma cell lines, it suggests that the oxygen sensing pathway plays a general role in controlling cell proliferation through upregulating Cav-1. Cav-1 should be considered as a target for drug design as the knock down of Cav-1 decreased cell proliferation and could potentially decrease tumour growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 459.

MDM2 is an E3 ligase that promotes ubiquitin-mediated destruction of p53. Cellular stresses such as DNA damage can lead to p53 activation due in part to MDM2 destabilization. Here, we show that the stability of MDM2 is regulated by an ubiquitin-like NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization concomitant with p53 activation. Concordantly, RNA-imediated knockdown of endogenous NEDP1 blocked diminution of MDM2 levels and increased chemoresistance of tumor cells. These findings unveil the regulation of MDM2 stability via NEDP1 as a common molecular determinant governing chemotherapy-induced p53-dependent cell death. Citation Information: Cancer Res 2009;69(23 Suppl):B1.