RAS and RHO family GTPase mutations in cancer: twin sons of different mothers?

Abstract

The RAS and RHO family comprise two major branches of the RAS superfamily of small GTPases. These proteins function as regulated molecular switches and control cytoplasmic signaling networks that regulate a diversity of cellular processes, including cell proliferation and cell migration. In the early 1980s, mutationally activated RAS genes encoding KRAS, HRAS and NRAS were discovered in human cancer and now comprise the most frequently mutated oncogene family in cancer. Only recently, exome sequencing studies identified cancer-associated alterations in two RHO family GTPases, RAC1 and RHOA. RAS and RHO proteins share significant identity in their amino acid sequences, protein structure and biochemistry. Cancer-associated RAS mutant proteins harbor missense mutations that are found primarily at one of three mutational hotspots (G12, G13 and Q61) and have been identified as gain-of-function oncogenic alterations. Although these residues are conserved in RHO family proteins, the gain-of-function mutations found in RAC1 are found primarily at a distinct hotspot. Unexpectedly, the cancer-associated mutations found with RHOA are located at different hotspots than those found with RAS. Furthermore, since the RHOA mutations suggested a loss-of-function phenotype, it has been unclear whether RHOA functions as an oncogene or tumor suppressor in cancer development. Finally, whereas RAS mutations are found in a broad spectrum of cancer types, RHOA and RAC1 mutations occur in a highly restricted range of cancer types. In this review, we focus on RHOA missense mutations found in cancer and their role in driving tumorigenesis, with comparisons to cancer-associated mutations in RAC1 and RAS GTPases.     

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.     

Role of RHO family interacting cell polarization regulators (RIPORs) in health and disease: Recent advances and prospects

The RHO GTPase family has been suggested to play critical roles in cell growth, migration, and polarization. Regulators and effectors of RHO GTPases have been extensively explored in recent years. However, little attention has been given to RHO family interacting cell polarization regulators (RIPORs), a recently discovered protein family of RHO regulators. RIPOR proteins, namely, RIPOR1-3, bind directly to RHO proteins (A, B and C) via a RHO-binding motif and exert suppressive effects on RHO activity, thereby negatively influencing RHO-regulated cellular functions. In addition, RIPORs are phosphorylated by upstream protein kinases under chemokine stimulation, and this phosphorylation affects not only their subcellular localization but also their interaction with RHO proteins, altering the activation of RHO downstream targets and ultimately impacting cell polarity and migration. In this review, we provide an overview of recent studies on the function of RIPOR proteins in regulating RHO-dependent directional movement in immune responses and other pathophysiological functions.     

The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy

Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family.