[Show abstract][Hide abstract] ABSTRACT: RAF inhibitors achieve unprecedented but mainly transient clinical responses in melanoma patients whose tumors harbor an activating BRAF mutation. One notable side-effect of RAF inhibitors is the stimulation of cutaneous skin tumors, arising in about 30% of patients receiving these drugs, which are thought to develop as a result of inhibitor-induced activation of wild-type Raf in occult precursor skin lesions. This effect raises the possibility that less manageable tumors might also arise in other epithelial tissues. Here we provide preclinical evidence supporting this disquieting hypothesis by showing that the RAF inhibitors PLX-4032 (vemurafenib) and GDC-0879 precipitate the development of cell-autonomous, Ras-driven tumors in skin and gastric epithelia. The magnitude of the effects correlated with the inhibitors' relative abilities to induce ERK activation. Epidermis-restricted ablation of either B-Raf or C-Raf prevented PLX-4032-induced ERK activation and tumorigenesis. In contrast, GDC-0879 induced ERK activation and tumorigenesis in B-Raf-deficient epidermis, whereas C-Raf ablation blocked GDC-0879-induced tumorigenesis (despite strong ERK activation) by preventing Rok-α-mediated keratinocyte de-differentiation. Thus, inhibitor-induced ERK activation did not require a specific Raf kinase. ERK activation was necessary, but not sufficient for Ras + Raf inhibitor-induced tumorigenesis, while C-Raf down-regulation of Rok-α was essential even in the face of sustained ERK signaling to prevent differentiation and promote tumorigenesis. Taken together, our findings suggest that combination therapies targeting ERK-dependent and ERK-independent functions of Raf may be more efficient but also safer for cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.
No preview · Article · Feb 2013 · Cellular Signalling
[Show abstract][Hide abstract] ABSTRACT: Ras-driven tumorigenesis is assumed to depend on Raf for ERK activation and proliferation; yet, an in vivo requirement for Raf as MEK/ERK activator in this setting has not been demonstrated to date. Here, we show that epidermis-restricted B-Raf ablation restrains the onset and stops the progression of established Ras-driven tumors by limiting MEK/ERK activation and proliferation. Concomitant elimination of B-Raf and Raf-1 enforces the abrupt regression of established tumors owing to the decrease in ERK activation and proliferation caused by B-Raf ablation combined with the ERK-independent increase in Rho-dependent kinase (Rok) signaling and differentiation triggered by Raf-1 inactivation. Thus, B-Raf and Raf-1 have non-redundant functions in Ras-driven tumorigenesis. Of note, Raf kinase inhibitors achieve impressive results in melanomas harboring oncogenic BRAF, but are ineffective against Ras-driven tumors; moreover, therapy-related skin tumors driven by a paradox ERK activation as well as primary and acquired resistance have been reported. Our results suggest that therapies targeting both Raf kinase-dependent and -independent pathways may be effective against a broader range of malignancies and reduce the risks of adverse effects and/or resistance.Oncogene advance online publication, 25 June 2012; doi:10.1038/onc.2012.254.
[Show abstract][Hide abstract] ABSTRACT: The epidermis is the outermost layer of the body and protects it from environmental insults. This crucial function is sustained by a continuous process of self-renewal involving the carefully balanced proliferation and differentiation of progenitor cells constantly replacing the mature cells at the surface of the epidermis. Genetic changes in the signalling pathways controlling keratinocyte proliferation and differentiation disrupt this balance and lead to pathological changes including carcinogenesis. This review discusses the role of Ras, an oncogene critically involved in the development of skin neoplasia, and its downstream effector Raf in epidermal homeostasis and tumourigenesis. In particular, we will focus on the recently established role of Raf-1 as the decisive element that, by restraining keratinocyte differentiation, allows the development and maintenance of Ras-driven tumours.
Full-text · Article · Mar 2011 · British Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Human papillomaviruses (HPV) of the genus β are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of β-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell-autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3(+/-):HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3(+/+):HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3(+/-):HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer.
[Show abstract][Hide abstract] ABSTRACT: The activity of Raf-1 and Rok-alpha kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-alpha, but the underlying mechanism is unknown. In this study, we show that Rok-alpha inhibition by Raf-1 relies on an intermolecular interaction between the Rok-alpha kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-alpha's own autoinhibitory region. Thus, Raf-1 mediates Rok-alpha inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-alpha-dependent defects of Raf-1-deficient cells. Downstream of Ras and Rho, the Raf-1-Rok-alpha interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.
Full-text · Article · Nov 2009 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Ras activation is common to many human cancers and promotes cell proliferation and survival by initiating multiple signaling cascades. Accordingly, Ras-transformed cells are generally considered too resourceful to become addicted to a single effector. In contrast to this tenet, we now demonstrate an absolute, cell autonomous requirement for Raf-1 in the development and maintenance of Ras-induced skin epidermis tumors. Mechanistically, Raf-1 functions as an endogenous inhibitor dimming the activity of the Rho-dependent kinase Rok-alpha in the context of a Ras-induced Raf-1:Rok-alpha complex. Raf-1-induced Rok-alpha inhibition allows the phosphorylation of STAT3 and Myc expression and promotes dedifferentiation in Ras-induced tumors. These data link the Raf-1:Rok-alpha complex to STAT3/Myc activation and delineate a pathway crucial for cell fate decision in Ras-induced tumorigenesis.