Aleksandra Franovic

Silencing of Epidermal Growth Factor Receptor Suppresses Hypoxia-Inducible Factor-2–Driven VHL−/− Renal Cancer

Inactivating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are associated with clear cell renal cell carcinoma (VHL-/- RCC), the most frequent malignancy of the human kidney. The VHL protein targets the alpha subunits of hypoxia-inducible factor (HIF) transcription factor for ubiquitination and degradation. VHL-/- RCC cells fail to degrade HIF resulting in the constitutive activation of its target genes, a process that is required for tumorigenesis. We recently reported that HIF activates the transforming growth factor-alpha/epidermal growth factor receptor (TGF-alpha/EGFR) pathway in VHL-defective RCC cells. Here, we show that short hairpin RNA (shRNA)-mediated inhibition of EGFR is sufficient to abolish HIF-dependent tumorigenesis in multiple VHL-/- RCC cell lines. The 2alpha form of HIF (HIF-2alpha), but not HIF-1alpha, drives in vitro and in vivo tumorigenesis of VHL-/- RCC cells by specifically activating the TGF-alpha/EGFR pathway. Transient incubation of VHL-/- RCC cell lines with small interfering RNA directed against EGFR prevents autonomous growth in two-dimensional culture as well as the ability of these cells to form dense spheroids in a three-dimensional in vitro tumor assay. Stable expression of shRNA against EGFR does not alter characteristics associated with VHL loss including constitutive production of HIF targets and defects in fibronectin deposition. In spite of this, silencing of EGFR efficiently abolishes in vivo tumor growth of VHL loss RCC cells. These data identify EGFR as a critical determinant of HIF-2alpha-dependent tumorigenesis and show at the molecular level that EGFR remains a credible target for therapeutic strategies against VHL-/- renal carcinoma.

Hypoxia Inducible Factor Activates the Transforming Growth Factor-α/Epidermal Growth Factor Receptor Growth Stimulatory Pathway in VHL-/- Renal Cell Carcinoma Cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL-/- RCC). VHL-/- RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL-/- RCC cells by activating the transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL-/- RCC cells. TGF-α is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL-/- RCC cells is a direct consequence of HIF activation. In contrast to TGF-α, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL-/- RCC cells. VHL-/- RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-α and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.

Translational up-regulation of the EGFR by tumor hypoxia provides a nonmutational explanation for its overexpression in human cancer

Overexpression of the EGF receptor (EGFR) is a recurrent theme in human cancer and is thought to cause aggressive phenotypes and resistance to standard therapy. There has, thus, been a concerted effort in identifying EGFR gene mutations to explain misregulation of EGFR expression as well as differential sensitivity to anti-EGFR drugs. However, such genetic alterations have proven to be rare occurrences in most types of cancer, suggesting the existence of a more general physiological trigger for aberrant EGFR expression. Here, we provide evidence that overexpression of wild-type EGFR can be induced by the hypoxic microenvironment and activation of hypoxia-inducible factor 2-α (HIF2α) in the core of solid tumors. Our data suggest that hypoxia/HIF2α activation represents a common mechanism for EGFR overexpression by increasing EGFR mRNA translation, thereby diminishing the necessity for gene mutations. This allows for the accumulation of elevated EGFR levels, increasing its availability for the autocrine signaling required for tumor cell growth autonomy. Taken together, our findings provide a nonmutational explanation for EGFR overexpression in human tumors and highlight a role for HIF2α activation in the regulation of EGFR protein synthesis.

An integrin β 3 –KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition

Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

An oxygen-regulated switch in the protein synthesis machinery

Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7-methylguanosine (m7-GpppG) 5′ cap of messenger RNAs. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms. Although the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. We show that hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA-binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array of mRNAs, including that encoding the epidermal growth factor receptor. Once assembled at the rHRE, the HIF-2α–RBM4–eIF4E2 complex captures the 5′ cap and targets mRNAs to polysomes for active translation, thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program by which oxygen tension switches the basic translation initiation machinery.

Human cancers converge at the HIF-2α oncogenic axis

Cancer development is a multistep process, driven by a series of genetic and environmental alterations, that endows cells with a set of hallmark traits required for tumorigenesis. It is broadly accepted that growth signal autonomy, the first hallmark of malignancies, can be acquired through multiple genetic mutations that activate an array of complex, cancer-specific growth circuits [Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70; Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789–799]. The superfluous nature of these pathways is thought to severely limit therapeutic approaches targeting tumor proliferation, and it has been suggested that this strategy be abandoned in favor of inhibiting more systemic hallmarks, including angiogenesis (Ellis LM, Hicklin DJ (2008) VEGF-targeted therapy: Mechanisms of anti-tumor activity. Nat Rev Cancer 8:579–591; Stommel JM, et al. (2007) Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 318:287–290; Kerbel R, Folkman J (2002) Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2:727–739; Kaiser J (2008) Cancer genetics: A detailed genetic portrait of the deadliest human cancers. Science 321:1280–1281]. Here, we report the unexpected observation that genetically diverse cancers converge at a common and obligatory growth axis instigated by HIF-2α, an element of the oxygen-sensing machinery. Inhibition of HIF-2α prevents the in vivo growth and tumorigenesis of highly aggressive glioblastoma, colorectal, and non–small-cell lung carcinomas and the in vitro autonomous proliferation of several others, regardless of their mutational status and tissue of origin. The concomitant deactivation of select receptor tyrosine kinases, including the EGFR and IGF1R, as well as downstream ERK/Akt signaling, suggests that HIF-2α exerts its proliferative effects by endorsing these major pathways. Consistently, silencing these receptors phenocopies the loss of HIF-2α oncogenic activity, abrogating the serum-independent growth of human cancer cells in culture. Based on these data, we propose an alternative to the predominant view that cancers exploit independent autonomous growth pathways and reveal HIF-2α as a potentially universal culprit in promoting the persistent proliferation of neoplastic cells.

Multiple Acquired Renal Carcinoma Tumor Capabilities Abolished upon Silencing of ADAM17

Malignancy is a manifestation of acquired defects in regulatory circuits that direct normal cell proliferation and homeostasis. Most of these circuits operate through cell autonomous pathways, whereas others potentially involve the neighboring microenvironment. We report that the metalloprotease ADAM17 plays a pivotal role in several acquired tumor cell capabilities by mediating the availability of soluble transforming growth factor-alpha, an epidermal growth factor receptor (EGFR) ligand, and thus the establishment of a key autocrine signaling pathway. Silencing of ADAM17 in human renal carcinoma cell lines corrects critical features associated with cancer cells, including growth autonomy, tumor inflammation, and tissue invasion. Highly malignant renal carcinoma cancer cells fail to form in vivo tumors in the absence of ADAM17, confirming the essential function of this molecule in tumorigenesis. These data show that ligand shedding is a crucial step in endogenous EGFR activation and endorse prospective therapeutic strategies targeting ADAM17 in human cancer.

A MEK-independent role for CRAF in mitosis and tumor progression.

RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia

Significance DNA methyltransferase 3a (DNMT3a) mediates the de novo methylation of DNA to regulate gene expression and maintain cellular homeostasis. Mutations in DNMT3a in primary tumors suggest that the DNMT3a epigenetic program is modified during early tumorigenesis. We show that a major consequence of DNMT3a defects is the epigenetic deregulation and unscheduled activation of the EPAS1 (hypoxia-inducible factor 2α) gene that facilitates growth and viability under conditions of low oxygen availability. This represents a critical step during tumorigenesis, because cancer cells must adapt to hypoxia during the formation of the earliest multicellular foci. The data identify the DNMT3a epigenetic program as a gatekeeper of the hypoxic cancer cell phenotype. Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.

ETS-1 Oncogenic Activity Mediated by Transforming Growth Factor α

Inappropriate expression of Ets-1 is observed in a variety of human cancers, and its forced expression in cultured cells results in transformation, autonomous proliferation, and tumor formation. The basis by which Ets-1 confers autonomous growth, one of the primary hallmarks of cancer cells and a critical component of persistent proliferation, has yet to be fully explained. Using a variety of cancer cell lines, we show that inhibition of Ets-1 blocks tumor formation and cell proliferation in vivo and autonomous growth in culture. A screen of multiple diffusible growth factors revealed that inhibition of Ets-1 results in the specific downregulation of transforming growth factor alpha (TGFalpha), the proximal promoter region of which contains multiple ETS family DNA binding sites that can be directly bound and regulated by Ets-1. Notably, rescuing TGFalpha expression in Ets-1-silenced cells was sufficient to restore tumor cell proliferation in vivo and autonomous growth in culture. These results reveal a previously unrecognized mechanism by which Ets-1 oncogenic activity can be explained in human cancer through its ability to regulate the important cellular mitogen TGFalpha.