Nawal Bendris

A novel function for Cyclin A2: Control of cell invasion via RhoA signaling

Cyclin A2 promotes RhoA activation, which inhibits cytoskeletal rearrangements and cell migration.

Cell cycle, cytoskeleton dynamics and beyond: the many functions of cyclins and CDK inhibitors.

While targeting experiments carried out on the genes encoding many cell cycle regulators have challenged our views of cell cycle control, they also suggest that redundancy might not be the only explanation for the observed perplexing phenotypes. Indeed, several observations hint at functions of cyclins and CDK inhibitors that cannot be accounted for by their sole role as kinase regulators. They are found involved in many cellular transactions, depending or not on CDKs that are not directly linked to cell cycle control, but participating to general mechanisms such as transcription, DNA repair or cytoskeleton dynamics. In this review we discuss the roles that these alternative functions might have in cancer cell proliferation and migration that sometime even challenge their definition as proliferation markers.

Cyclin A2, Rho GTPases and EMT

Cell cycle regulators, such as cyclins, are often upregulated in many proliferative disorders, and Cyclin A2 is generally considered as a marker of aggressive cancers. Our recent work, which revealed decreased expression of Cyclin A2 upon metastasis of colorectal cancer, suggests a more complicated situation. Consistent with this, we identified a role for Cyclin A2, via RhoA, in regulation of the actin cytoskeleton and the control of cell invasion. Cyclin A2 also regulates spindle orientation which, when misoriented, could disrupt cell polarity and favor cancer cell detachment from the tumor as part of a transforming process, such as epithelial to mesenchymal transition (EMT). During EMT, cells undergo morphological and molecular changes toward a mesenchymal phenotype. Upregulation, or increased activity of some Rho GTPases, such as Cdc42, Rac1 or RhoC, increases the invasive potential of these cells. This correlates with the inverse relationship between RhoA and RhoC activities we observed in an epithelial cell type. Altogether, these observations raise the possibility that Cyclin A2 is instrumental in preventing EMT and therefore cancers of epithelial tissues.

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Sorting nexin 9 is a multifunctional scaffold protein that coordinates endocytic trafficking, activation of RhoGTPases, and actin nucleation via N-WASP to modulate cancer cell invasion and metastasis.

Endocytosis, Metastasis and Beyond: Multiple Facets of SNX9

Sorting nexin (SNX)9 was first discovered as an endocytic accessory protein involved in clathrin-mediated endocytosis. However, recent data suggest that SNX9 is a multifunctional scaffold that coordinates membrane trafficking and remodeling with changes in actin dynamics to affect diverse cellular processes. Here, we review the accumulated knowledge on SNX9 with an emphasis on its recently identified roles in clathrin-independent endocytic pathways, cell invasion, and cell division, which have implications for SNX9 function in human disease, including cancer.

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

Significance Clathrin-mediated endocytosis (CME) regulates receptor trafficking, thereby affecting several cellular signaling pathways. We discovered that dynamin-1 is selectively activated downstream of TNF-related apoptosis-inducing ligand–death receptors (TRAIL–DRs) to self-regulate their endocytosis, attenuate apoptotic signaling, and increase cell survival. Activation of initiator caspase-8 by TRAIL–DRs triggers spikes of Ca2+ through ryanodine receptor calcium channels, activating calcineurin, and in turn dephosphorylating dynamin-1 to promote cargo-selective endocytosis of TRAIL–DR. This study delineates specific mechanisms linking signaling downstream of cell-surface receptors to the regulation of cargo-selective CME, and thus their signaling properties. Cancer cell-specific adaptation of this bidirectional crosstalk between signaling and CME has implications for tumor progression and metastasis. Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

A Systematic Analysis Reveals Heterogeneous Changes in the Endocytic Activities of Cancer Cells

Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics.

Cyclin A2, a novel regulator of EMT

Our previous work showed that Cyclin A2 deficiency promotes cell invasion in fibroblasts. Given that the majority of cancers emerge from epithelia, we explored novel functions for Cyclin A2 by depleting it in normal mammary epithelial cells. This caused an epithelial to mesenchymal transition (EMT) associated with loss of cell-to-cell contacts, decreased E-Cadherin expression and increased invasive properties characterized by a reciprocal regulation of RhoA and RhoC activities, where RhoA-decreased activity drove cell invasiveness and E-Cadherin delocalization, and RhoC-increased activity only supported cell motility. Phenotypes induced by Cyclin A2 deficiency were exacerbated upon oncogenic activated-Ras expression, which led to an increased expression of EMT-related transcriptional factors. Moreover, Cyclin A2-depleted cells exhibited stem cell-like properties and increased invasion in an in vivo avian embryo model. Our work supports a model where Cyclin A2 downregulation facilitates cancer cell EMT and metastatic dissemination.

Sorting nexin 9 negatively regulates invadopodia formation and function in cancer cells

ABSTRACT The ability of cancer cells to degrade the extracellular matrix and invade interstitial tissues contributes to their metastatic potential. We recently showed that overexpression of sorting nexin 9 (SNX9) leads to increased cell invasion and metastasis in animal models, which correlates with increased SNX9 protein expression in metastases from human mammary cancers. Here, we report that SNX9 expression is reduced relative to neighboring normal tissues in primary breast tumors, and progressively reduced in more aggressive stages of non-small-cell lung cancers. We show that SNX9 is localized at invadopodia where it directly binds the invadopodia marker TKS5 and negatively regulates invadopodia formation and function. SNX9 depletion increases invadopodia number and the local recruitment of MT1-MMP by decreasing its internalization. Together, these effects result in increased localized matrix degradation. We further identify SNX9 as a Src kinase substrate and show that this phosphorylation is important for SNX9 activity in regulating cell invasion, but is dispensable for its function in regulating invadopodia. The diversified changes associated with SNX9 expression in cancer highlight its importance as a central regulator of cancer cell behavior. Summary: SNX9, differentially regulated by Src, alters the ability of cancer cells to degrade the matrix through regulation of MT1-MMP internalization and/or invadopodia formation and function.

Cyclin A2 modulates EMT via β-catenin and phospholipase C pathways

We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that β-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of β-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.