Targeted therapy for the loss of von hippel-lindau in renal cell carcinoma

Abstract

Radiation and conventional cytotoxic chemotherapies are ineffective in treating renal cancer. Approximately 75 percent of renal cell carcinoma (RCC) is associated with an inactivation of the tumor suppressor gene von Hippel-Lindau (VHL). We exploited the possibility of targeting VHL-deficient RCC through synthetic lethality using a high-throughput screening approach. In this screen, STF-62247 was identified to be selectively toxic and growth inhibitory to renal cells lacking VHL. We recently demonstrated that the cytotoxicity of STF-62247 is due to dysregulated autophagy. Furthermore, the reduction of protein levels of essential autophagy pathway components such as Atg5, Atg7 and Atg9 reduces sensitivity of VHL-deficient cells to killing by STF-62247. Loss of proteins involved in Golgi trafficking sensitized RCC with wild-type VHL to killing by STF-62247, indicating a potential role for these proteins as a target of the compound. Our study supports the concept of using synthetic lethality to selectively kill VHL-deficient cells that represents a new type of targeted therapy for the treatment of RCC.

Targeted Therapy for Synthetic Lethality as a New Form of Treatment for RCC

Renal cell carcinoma (RCC) is one of the most lethal of all urological cancers with approximately 36,000 new diagnoses reported in the United States every year that result in 13,000 deaths.¹ RCC is often associated with metastatic disease and effective systemic therapies do not exist to treat kidney cancer. Radiation, immunotherapy and chemotherapy have been reported to slightly increase survival, but most RCC tumors are refractory to these standard therapies and ultimately progress to the metastatic state. Targeted genetic approaches have been investigated for their efficacy in treating RCC.² For example, different agents that can inhibit the hypoxia inducible factor (HIF) pathway are presently in clinical trials: temsirolimus and everolimus inhibit the mTOR pathway and affect HIF translation; soratenib, sunitinib and bevacizumab inhibit vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) signaling, and VEGF ligand binding to VEGFR, respectively, and possess antiangiogenic activity; erbitux inhibits binding of EGF to its receptor and decreases tumor cell proliferation. All of these therapies have shown some effectiveness in the management of renal cell cancer to different degrees.^3,4 In contrast to these targeted therapies, we proposed the concept of a new type of targeted therapy based on the idea of synthetic lethality as an approach to selectively kill RCC cells.^5,6 Synthetic lethality occurs when two nonallelic mutations, which by themselves are not lethal, result in cell death when combined. Inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) occurs in 75 percent of sporadic renal cell carcinomas and is an early event that promotes tumorigenesis, leading to clear cell-renal cell cancer.⁷ In addition, germ-line mutations of VHL are found in patients with VHL disease. These patients develop hemangioblastomas of the retina and the CNS, renal cysts and carcinomas, pancreatic cysts and tumors and pheochromocytomas.⁸ In this regard, targeting the loss of the von Hippel-Lindau tumor suppressor gene by synthetic lethality has promising implications in the development of new therapies for renal cell carcinoma.

Autophagy is Induced by STF-62247 Treatment

Cell-based small molecule screening has been used to discover compounds that inhibit specific proteins, such as HIF, or to overcome drug resistance to reduce tumorigenicity.^9-12 Recently, we used a chemical library of diverse small molecules to perform a fluorescence-based mammalian screen in wild-type VHL and VHL-deficient RCCs that were stably transfected with EYFP (manuscript in preparation). The selective toxicity to the absence of VHL of the small molecules identified from this synthetic lethal screen was validated by colony survival assays. Among them, STF-62247 demonstrated an inhibitory concentration, IC₅₀, that is 25-fold lower in VHL-deficient cells compared to their genetic counterpart wild-type VHL cells.⁶ The selective cytotoxicity of STF-62247 for the loss of VHL is observed in different renal carcinoma cells and inhibits the growth of tumors that lack VHL. The best documented role for pVHL, the protein product of the VHL tumor suppressor gene, is its E3-ubiquitin ligase activity towards HIFα.^13,14 Interestingly, we found that the selective toxicity of STF-62247 in cells lacking VHL occurs through a HIF-independent mechanism.

In characterizing the mechanism of cell death induced by STF-62247, we did not detect apoptosis or DNA damage in response to STF-62247 in either VHL wild-type or -deficient RCCs. However, STF-62247 induced the formation of large intracytoplasmic vacuoles, characteristic of cells undergoing autophagy. Autophagy is a lysosomal degradation pathway that regulates the turnover of organelles and long-lived proteins and is essential for survival, development and differentiation.¹⁵ Under certain conditions, autophagy has been proposed to act as a protective pathway against cancer, aging, neurodegeneration and heart disease, but persistent stress or prolonged starvation can also result in autophagic cell death.¹⁶ What signals are responsible for autophagy to either protect against or cause cell death are not well understood.

Under metabolic stress, an autophagosome sequesters portions of the cytoplasm and organelles in a double-membrane vesicle that fuses with a lysosome to become an autolysosome where hydrolases degrade their contents.¹⁷ The elongation of the double-membrane involves two ubiquitin-like conjugation systems: the Atg12-Atg5 complex and the mammalian LC3 or yeast Atg8 protein.^18,19 We monitored STF-62247-induced autophagy by an increase in the LC3-lipidated form, a processed nonsoluble form of LC3 that is associated with the phagophore and the autophagosomal membrane of cells undergoing autophagy. Furthermore, STF-62247 double-membrane vesicles are also observed by electron microscopy. To determine whether autophagy induced by STF-62247 was responsible for cytotoxicity of VHL-deficient cells, we measured cell survival in response to STF-62247 following inhibition of essential autophagy components. Silencing the autophagy-related genes Atg5, Atg7 or Atg9 by siRNA increases cell survival in VHL-deficient cells in response to STF-62247, confirming that the induction of autophagy by STF-62247 in cells lacking functional VHL results in cell death. Atg7 acts as an E1 enzyme in the autophagy conjugation system, whereas Atg9 cycles between the trans-Golgi network and endosomes.^20,21 Both of these genes act upstream of LC3 processing and play a role in the formation of the double-membrane vesicle. Beclin 1 (BECN), the mammalian ortholog of yeast Atg6, acts as a tumor suppressor gene in mice and is frequently deleted in human cancers. In mammalian cells, Beclin 1 forms a complex with Vps34, Vps15 and UVRAG, and the antiapoptotic proteins Bcl-2/Bcl-X_L can also bind Beclin 1.¹⁵ Preliminary results indicate that STF-62247-induced selective toxicity is not affected by silencing Beclin 1 but further studies need to be addressed.

Although the induction of large vacuoles and LC3 processing is observed in VHL-deficient cells and could be associated with STF-62247-toxicity, the increase of LC3 was also observed in wild-type VHL cells, and suggests that the presence of autophagosomes cannot by themselves explain the difference in toxicity between RCC cells with and without VHL. It is noteworthy that VHL-deficient cells exhibited higher acidification of mature autolysosomes in response to STF-62247, compared to their wild-type counterparts. Staining with monodansylcadaverine (MDC), which labels some of the acidic compartments that are observed after fusion with lysosomes, was primarily detected around larger vacuoles compared to the punctate staining of LC3, suggesting that these large vacuoles are associated with the acidic components of autolysosomes. MDC staining induced by STF-62247 was attenuated using NH₄Cl, which also blocked vesicle acidification, and suggests that the fusion of autophagosomes with lysosomes is a key step leading to autophagic cell death in VHL-deficient cells.

Using the yeast deletion pool to understand the mechanism of STF-62247 killing, we showed that Golgi trafficking is a target of STF-62247. Recent studies suggest a role for the Golgi and vacuolar protein-sorting in autophagy and autophagosome formation.^21,22 Reduced levels of ALG5, OSBP3 or CHMP6, which are all regulated by VHL are involved in coordinating vesicular transport between the ER, the trans-Golgi network and lysosomes, sensitized VHL cells to STF-62247. In addition, knockdown of these proteins using siRNA increased vacuole formation in response to STF-62247, strongly implicating the trans-Golgi network as a critical pathway linked to STF-62247 cytotoxicity.

Our recent paper identified a novel small molecule that targets the loss of the tumor suppressor gene von Hippel-Lindau through synthetic lethality.⁶ We show that STF-62247 induces autophagic cell death in VHL-deficient cells compared to wild-type VHL cells and suggests that, in addition to its well-understood role in HIF degradation, VHL has other critical targets that also play important roles in autophagy (Fig. 1).

Figure 1.

Effect of the STF-62247 in renal cell carcinomas through autophagy. STF-62247 disrupts ER-Golgi trafficking through CHMP6, OSBPL3 and ALG5, and induces autophagy. Lipidation associated with the formation of the double-membrane vesicle is observed in an Atg5-, 7- and 9-dependent manner. Higher acidification of autolysosomes is correlated with autophagic cell death in VHL-deficient cells, whereas wild-type VHL cells are protected against acidification and survive. This figure is adapted from Turcotte et al., Cancer Cell, 14 July 2008.

Future Directions

Discovery of new therapeutic compounds for clinical management of tumors has been possible due to a better understanding of the genetics, biology and molecular biology of the cancer cell. The possibility of targeting the loss of a tumor suppressor gene or the overexpression of an oncogene through synthetic lethality has promising implications for the treatment of cancer. Increasing evidence indicates a role for autophagy in cancer. How autophagic cells can promote survival or induce cell death requires further investigation, but anticancer therapies such as radiation, tamoxifen, rapamycin, imatinib, arsenic trioxide or resveratrol have all been identified to induce autophagic cell death. We identified a small molecule that induces autophagic cell death selectively in VHL-deficient RCC and demonstrate the possibility of targeted therapy by synthetic lethality in RCC that could serve as a paradigm for other solid tumors.

Abbreviations

Atg

autophagy-related gene

CHMP6

charged multivesicular body protein 6

EGFR

epidermal growth factor receptor

HIF

hypoxia inducible factor

LAMP1 or 2

lysosome-associated membrane protein 1 or 2

MAP-LC3

microtubule associated protein light chain 3

3-MA

3-methyladenine

OSBPL3

oxysterol binding protein-like 3

PDGF

platelet-derived growth factor

PI3K

phosphatidylinositol 3-kinase

RCCs

renal cell carcinomas

TGFα

transforming growth factor-α

TGN

trans golgi network

VEGF

vascular endothelial growth factor

VHL

von Hippel-Lindau