RHOB expression controls the activity of serine/threonine protein phosphatase PP2A to modulate mesenchymal phenotype and invasion in non-small cell lung cancers

ABSTRACT

Metastatic dissemination is the cause of death in the vast majority of cancers, including lung cancers. In order to metastasize, tumor cells must undergo a well-known series of changes, however the molecular details of how they manage to overcome the barriers at each stage remain incomplete. One critical step is acquiring the ability to migrate through the extracellular matrix. Loss of expression of the RAS-related small GTPase RHOB is a common feature of lung cancer progression, and we recently reported that this induces an epithelial-to-mesenchymal transition (EMT) that is dependent on SLUG overexpression and E-Cadherin inhibition and is characterized by 3-dimensional cell shape reorganization and the increased invasiveness of bronchial cells. RHOB loss was found to induce AKT1 activation, which in turn activates RAC1 through its GEF TRIO. Further investigation of this pathway revealed that RHOB interacts with and positively regulates PP2A, one of the major cellular serine-threonine phosphatases, by recruiting its regulatory subunit B55. Here we discuss the role of this newly discovered RHOB/PP2A/AKT1/RAC1 pathway in relation to mesenchymal migration and invasion in lung cancer.

Introduction

Despite the emergence of novel effective therapies, lung cancer continues to be responsible for the greatest number of cancer-related deaths worldwide,¹ mainly due to its high rate of metastatic dissemination. The process by which tumor cells become metastatic involves many steps, a critical one being the acquisition of the ability to escape from the primary tumor and invade the proximal tissue. This phenotypic change is orchestrated by highly regulated cellular machinery, implying that complex molecular rearrangements within non-invasive cancer cells are required in order to migrate through the extracellular matrix. One of the earliest steps in the metastatic process is the epithelial to mesenchymal transition (EMT), during which cells lose epithelial cell junction proteins (e.g. E-cadherin) and gain mesenchymal markers such as N-cadherin, vimentin or fibronectin.² There are 2 main modes of 3-dimensional (3D) migration in cancer cells, defined by the morphological characteristics of the cell: i) mesenchymal migration, in which cells adopt a “fibroblast-like” elongated shape; this is dependent upon extracellular matrix degradation via the secretion of active matrix metalloproteases (MMPs), and ii) amoeboid migration, characterized by a rounded cell shape; these cells can “glide” through matrix barriers via squeezing movements so often this does not require matrix proteolysis. The majority of tumor cells move via mesenchymal migration, and the mechanisms underlying this are predominantly initialized by EMT transcription factors (EMT-TFs) such as SNAIL, ZEB-1 or SLUG which modulate gene expression to favor mesenchymal transformation.² The different processes involved in EMT transition are often packaged together as a single entity but are actually regulated separately by many different actors and involve different signaling pathways. One of these pathways facilitates cell morphological reorganization through cytoskeletal rearrangements and is tightly regulated by several proteins that belong to the RHO GTPase sub-family.

RHO family proteins are critical in determining the type of cell migration

The Rho GTPase family is comprised of 20 members in humans, of which RHO, RAC1 and CDC42 remain the best studied. They are known to be involved in multiple cellular processes including cytoskeletal organization, cell motility, cell adhesion and transcription. Once activated by their guanine nucleotide exchange factors (GEFs), the RHO GTPases can bind to several effectors (mainly kinases and actin-binding partners) and directly regulate the assembly or disassembly of filamentous (F)-actin. CDC42 is involved in the extension of filopodia, RHOA mediates stress fiber formation, and RAC is involved in the formation of lamellipodia.³ In terms of types of migration, RHOA regulates contractile amoeboid migration via the ROCK-myosin II signaling axis,⁴ whereas CDC42 and RAC are thought to control mesenchymal migration through the WAVE and WASP proteins.⁵ Despite their high degree of homology, RHOA, RHOB and RHOC display different effects on cell migration and invasion, and this is also dependent on the cell type.⁶ RHOB, unlike RHOA and RHOC, is often downregulated in human tumors and is considered to behave as a tumor suppressor.^7-9 Its expression is decreased in highly invasive and poorly differentiated carcinoma, and the loss of RHOB correlates with lung tumor progression.^10-14 Recently, it was shown that not only RHOB is a strong prognostic factor in non-small cell lung cancers (NSCLC), but that it is also critical for the acquisition of an aggressive adenocarcinoma phenotype.¹⁵ Moreover, inhibition of RHOB was shown to promote migration and invasion of bronchial cells both in vitro and in vivo via an AKT-dependent mechanism.¹⁶ Recently, we shed light on a new signaling pathway that links the loss of RHOB with the invasion of bronchial cells (discussed in further detail below).¹⁷ These findings have improved our understanding of the tumor suppressor behavior of RHOB and its disruption in lung cancer.

Low RHOB expression induces a SLUG-dependent mesenchymal phenotype

Studying cell invasion in vitro requires cells to be cultured in a 3D environment. A matrigel/collagen mixture is usually used to mimic the pulmonary extracellular matrix¹⁸ and allows to easily monitor invading cell behavior. In our recent study, several pulmonary cell lines were seeded at low density under these 3D conditions in order to ensure the complete separation of cells and to better appreciate their 3D morphologies. We observed that some cell lines adopted a rounded shape, others had a fibroblast-like elongated morphology and a third group displayed a mixed phenotype. Analysis of RHOB levels showed that RHOB expression was higher in rounded cells than in elongated cells, suggesting a possible relationship between RHOB loss, cell invasion and 3D cell morphology. RHOB inhibition consistently induced the elongation of several bronchial cells whereas RHOB overexpression increased the percentage of rounded cells. Thus, consistent with our previous observations showing an increased invasiveness of lung cells after RHOB inhibition,¹⁶ these results demonstrated that this was associated with a mesenchymal phenotype. To determine whether this morphological switch was due to an amoeboid-mesenchymal transition (AMT) or an EMT, we conducted an expression analysis of a panel of genes involved in cell migration. This showed that RHOB inhibition strongly increased the mRNA levels of the transcription factor SLUG and decreased E-cadherin mRNA and protein levels, 2 major markers of the mesenchymal phenotype, suggesting a role for RHOB in EMT rather than AMT. In a similar study in prostate cancer cells, Vega and colleagues also reported that RHOB depletion reduced E-cadherin expression,¹⁹ supporting a role for RHOB in the EMT process (Fig. 1). SLUG overexpression and RHOB downregulation have also been associated with poor survival in lung carcinoma and in other cancers.^20-23 A recent meta-analysis proposed that SLUG protein expression levels could be considered for use as a biomarker for poor prognosis in head and neck cancer, lung and urinary carcinomas.²⁴ RHOB downregulation is thought to affect not all but specific proteins implicated in EMT since SNAIL expression was not significantly modified in RHOB-deficient cells despite being closely related to SLUG. Several other studies have also reported the overexpression of SLUG but not SNAIL during EMT,^25,26 suggesting a unique function for SLUG within the family of EMT regulators.²⁷ Interestingly, in our model SLUG inhibition was sufficient to prevent the increased invasion and morphological changes mediated by RHOB downregulation. It is noteworthy that RHOB and SLUG were also shown to cooperate in determining the migratory capacity of neural crest cells during chicken embryogenesis,²⁸ suggesting that these proteins act together to control cell migration in very different processes.

Figure 1.

Effect of transformation-induced RHOB downregulation on AKT1/RAC1, SLUG and YAP activation in NSCLC.

RHOB downregulation activates the AKT1-TRIO-RAC1 signaling axis to promote tumor cell invasion

In a previous study, we demonstrated that RHOB inhibition induces AKT1 phosphorylation and RAC1 activation, however the nexus between these 2 pathways was not determined.¹⁶ In our recent study we used pharmacological inhibitors and specific siRNA knockdowns to establish that the loss of RHOB activates the signaling cascade AKT1-TRIO-RAC1 (Fig. 1). Specifically, when RHOB was downregulated, RAC1 activation was dependent on TRIO and not TIAM1, 2 major GEFs of RAC1. TRIO inhibition was sufficient to reverse the mesenchymal phenotype triggered by RHOB downregulation. TRIO was also recently implicated in the invasion process in colorectal and hepatocellular carcinoma.^29,30 But while it was recently reported that AKT phosphorylates TIAM1 and thereby increases its stability,³¹ nothing is currently known about the regulation of TRIO by AKT. Very recently, Marcos-Ramiro and colleagues also reported a relationship between RHOB and RAC1 in the endothelium, where RHOB was shown to negatively regulate RAC1 activity and trafficking at endosomes after injury, resulting in the inhibition of barrier recovery after acute contraction.³² These studies suggest that the regulation of RAC1 activity could be dependent on RHOB-dependent control of vesicle traffic, as has been described for AKT.³³

AKT appears to be a key partner of RHOB in the control of numerous cell functions,^34,35 notably in RHOB-mediated regulation of EMT and the invasion of bronchial cells. However, the mechanism by which RHOB regulates phosphorylated AKT levels in cells is yet to be elucidated. In our model, neither PI3K nor PTEN, 2 key regulators of AKT phosphorylation, were found to account for RHOB-induced regulation of AKT phosphorylation. However, dephosphorylation of AKT by the PP2A phosphatase is known to be an important mechanism in determining the cellular levels of phosphorylated AKT, and although it was shown that RHOB binds to the catalytic subunit of PP2A,³⁶ the consequences of this binding were previously unknown.

RHOB binds and activates the tumor suppressor PP2A

PP2A, one of the major serine/threonine phosphatases, is a trimeric holoenzyme containing a scaffold subunit (PP2A-A), a catalytic subunit (PP2A-C), and a regulatory B subunit.³⁷ Four B subunit families have been described: B (B55/PR55), B′ (B56/PR61), B″ (PR48/72/130) and B‴ (PR93/110). The type of B subunit bound to the PP2A dimer determines both the substrate specificity and the cellular localization of the PP2A holoenzyme complex.³⁸ PP2A controls a variety of cellular functions through a broad spectrum of substrates, including cell cycle regulation, apoptosis, mitosis and DNA damage repair, and controls major signaling pathways including the MAPK and AKT pathways.^39,40 Thus, decreased PP2A activity promotes cellular transformation, and numerous studies have highlighted a tumor suppressor role for PP2A.^41,42 PP2A loss-of-function occurs by molecular mechanisms such as mutations of the PP2A subunits, phosphorylation and methylation of PP2A-C,⁴³ deregulation of the expression of the regulatory subunits or endogenous PP2A inhibitors.^42,44

Our recent report showed that RHOB binds to and modulates PP2A activity by recruiting the B55 regulatory subunit. When RHOB is silenced, B55 can dissociate from PP2A-C, inactivating the phosphatase and thereby increasing the levels of phosphorylated AKT. Similarly, the loss of B55 expression increases the level of phosphorylated AKT^45,46 while its overexpression impairs AKT phosphorylation,⁴⁷ underlining a role for B55 in PP2A-mediated dephosphorylation of AKT. Furthermore, our study shows that regulation of PP2A by RHOB directly impacts on the EMT process and invasion.^17,48 This strong link between RHOB, PP2A and invasion was also highlighted in a recent study examining the role of the tumor suppressor protein RASSF1A (Ras-association domain family 1 isoform A) in bronchial cells,⁴⁹ in which the Hippo pathway scaffold RASSF1A was shown to be inactivated by promoter hypermethylation and was associated with poor prognosis in lung cancers.⁵⁰ RASSF1A downregulation was also shown to induce EMT and increase the invasive and metastatic properties of lung cancer cells in vivo.⁴⁹ Interestingly, we found that inhibition of RASSF1A inactivated both RHOB and PP2A, decreased PP2A-RhoB complex and induced the nuclear translocation of the transcriptional cofactor YAP to promote bronchial cell invasion (Fig. 1). Remarkably, decreased PP2A activity in RASSF1A-depleted cells was responsible for the increased Ser885-phosphorylation and inactivation of GEFH1 and the subsequent inactivation of RHOB (Fig. 1). This feedback loop reinforces the importance of the link between RHOB and PP2A in the control of cancer cell transformation and progression.

Further to these studies we have recently reported a new functional link between RHOB and PP2A.⁴⁸ PP2A is known to play a prevalent role in dephosphorylating Histone 2A (γH2AX) after treatment with the selective topoisomerase I inhibitor camptothecin.⁵¹ Moreover, PP2A inhibition impairs double strand break (DSB) repair by homologous recombination.⁵² Interestingly, our results showed that RHOB-deficient cells have reduced PP2A activity, fail to dephosphorylate γH2AX following camptothecin treatment and show reduced DSB repair by homologous recombination.

Conclusion

RHOB downregulation is a crucial event during lung cancer progression and induces EMT by increasing SLUG expression, decreasing E-Cadherin expression, and triggering activation of the AKT1/TRIO/RAC1 signaling axis (Fig. 1). These molecular changes are responsible for the increased cell migration and invasion of bronchial tumor cells. Our results shed light on a strong link between RHOB and the tumor suppressor PP2A and through the downregulation of RHOB expression we were able to provide new insights into the inhibitory effects of PP2A activity in cancer progression. Altogether, these recent findings confirm an association between RHOB and another tumor suppressor pathway that could directly impact on bronchial tumor cell plasticity and invasion.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.