Phase 2 Trial of the Farnesyltransferase Inhibitor Tipifarnib for Relapsed/Refractory Peripheral T Cell Lymphoma.

A phase 2, international, open-label, non-randomized, single-arm trial was conducted to evaluate the efficacy and safety of tipifarnib, a farnesyltransferase inhibitor, as monotherapy for relapsed/refractory peripheral T-cell lymphoma (PTCL) and to evaluate tumor mutation profile as a biomarker of response. Adults with relapsed/refractory PTCL received tipifarnib 300 mg orally twice daily for 21 days in a 28-day cycle. The primary endpoint was objective response rate (ORR); secondary endpoints included ORR, progression-free survival (PFS), duration of response (DOR), and adverse events (AEs) in specific subtypes. Sixty-five patients with PTCL were enrolled: n=38 angioimmunoblastic T-cell lymphoma (AITL), n=25 PTCL not otherwise specified (PTCL-NOS), and n=2 other T-cell lymphomas. The ORR was 39.7% (95% CI, 28.1-52.5) in all patients and 56.3% (95% CI, 39.3-71.8) for AITL. Median PFS was 3.5 months overall (954% CI, 2.1-4.4), and 3.6 months (95% CI, 1.9-8.3) for AITL. Median DOR was 3.7 months (95% CI, 2.0-15.3), and greatest in AITL patients (7.8 months; 95% CI, 2.0-16.3). The median overall survival was 32.8 months (95% CI, 14.4 to not applicable). Tipifarnib-related hematologic AEs were manageable and included: neutropenia (43.1%), thrombocytopenia (36.9%), and anemia (30.8%); other tipifarnib-related AEs included nausea (29.2%) and diarrhea (27.7%). One treatment-related death occurred. Mutations in RhoA, DNMT3A, and IDH2 were seen in 60%, 33%, and 27%, respectively, in the AITL tipifarnib responder group vs 36%, 9%, and 9% in the non-responder group. Tipifarnib monotherapy demonstrated encouraging clinical activity in heavily pre-treated relapsed/refractory PTCL, especially in AITL, with a manageable safety profile. ClinicalTrials.gov NCT02464228.

Glucocorticoid mediated inhibition of LKB1 mutant non-small cell lung cancers.

The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity (p < 10-16) but was not mechanistically involved in DEX sensitivity. Subcutaneous, orthotopic and metastatic NSCLC xenografts, biomarker-selected, STK11/LKB1 mutant patient derived xenografts, and genetically engineered mouse models with KRAS/LKB1 mutant lung adenocarcinomas all showed marked in vivo anti-tumor responses with the glucocorticoid dexamethasone as a single agent or in combination with cisplatin. Mechanistically, GR activation triggers G1/S cell cycle arrest in LKB1 mutant NSCLCs by inducing the expression of the cyclin-dependent kinase inhibitor, CDKN1C/p57(Kip2). All findings were confirmed with functional genomic experiments including CRISPR knockouts and exogenous expression. Importantly, DEX-GR mediated cell cycle arrest did not interfere with NSCLC radiotherapy, or platinum response in vitro or with platinum response in vivo. While DEX induced LKB1 mutant NSCLCs in vitro exhibit markers of cellular senescence and demonstrate impaired migration, in vivo DEX treatment of a patient derived xenograft (PDX) STK11/LKB1 mutant model resulted in expression of apoptosis markers. These findings identify a previously unknown GR mediated therapeutic vulnerability in STK11/LKB1 mutant NSCLCs caused by induction of p57(Kip2) expression with both STK11 mutation and high expression of CPS1 as precision medicine biomarkers of this vulnerability.

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties.

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514-24. ©2017 AACRSee related editorial by Hynds and Janes, p. 491.

Crosstalk between CLCb/Dyn1-Mediated Adaptive Clathrin-Mediated Endocytosis and Epidermal Growth Factor Receptor Signaling Increases Metastasis.

Signaling receptors are internalized and regulated by clathrin-mediated endocytosis (CME). Two clathrin light chain isoforms, CLCa and CLCb, are integral components of the endocytic machinery whose differential functions remain unknown. We report that CLCb is specifically upregulated in non-small-cell lung cancer (NSCLC) cells and is associated with poor patient prognosis. Engineered single CLCb-expressing NSCLC cells, as well as "switched" cells that predominantly express CLCb, exhibit increased rates of CME and altered clathrin-coated pit dynamics. This "adaptive CME" resulted from upregulation of dynamin-1 (Dyn1) and its activation through a positive feedback loop involving enhanced epidermal growth factor (EGF)-dependent Akt/GSK3ß phosphorylation. CLCb/Dyn1-dependent adaptive CME selectively altered EGF receptor trafficking, enhanced cell migration in vitro, and increased the metastatic efficiency of NSCLC cells in vivo. We define molecular mechanisms for adaptive CME in cancer cells and a role for the reciprocal crosstalk between signaling and CME in cancer progression.

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

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.

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.

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

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.

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

Despite current advances in cancer research, metastasis remains the leading factor in cancer-related deaths. Here, we identify sorting nexin 9 (SNX9) as a new regulator of breast cancer metastasis. We detected an increase in SNX9 expression in human breast cancer metastases compared with primary tumors and demonstrated that SNX9 expression in MDA-MB-231 breast cancer cells is necessary to maintain their ability to metastasize in a chick embryo model. Reciprocally, SNX9 knockdown impairs the process. In vitro studies using several cancer cell lines derived from a variety of human tumors revealed a role for SNX9 in cell invasion and identified mechanisms responsible for this novel function. We showed that SNX9 controls the activation of RhoA and Cdc42 GTPases and also regulates cell motility via the modulation of well-known molecules involved in metastasis, namely RhoA-ROCK and N-WASP. In addition, we have discovered that SNX9 is required for RhoGTPase-dependent, clathrin-independent endocytosis, and in this capacity, can functionally substitute to the bona fide Rho GAP, GRAF1 (GTPase Regulator Associated with Focal Adhesion Kinase). Together, our data establish novel roles for SNX9 as a multifunctional protein scaffold that regulates, and potentially coordinates, several cellular processes that together can enhance cancer cell metastasis.

Cyclin A2: At the crossroads of cell cycle and cell invasion.

Cyclin A2 is an essential regulator of the cell division cycle through the activation of kinases that participate to the regulation of S phase as well as the mitotic entry. However, whereas its degradation by the proteasome in mid mitosis was thought to be essential for mitosis to proceed, recent observations show that a small fraction of cyclin A2 persists beyond metaphase and is degraded by autophagy. Its implication in the control of cytoskeletal dynamics and cell movement has unveiled its role in the modulation of RhoA activity. Since this GTPase is involved in both cell rounding early in mitosis and later, in the formation of the cleavage furrow, this suggests that cyclin A2 is a novel actor in cytokinesis. Taken together, these data point to this cyclin as a potential mediator of cell-niche interactions whose dysregulation could be taken as a hallmark of metastasis.

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 modulates EMT via ß-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 ß-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 ß-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.

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, 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.

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.

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

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.

Cyclin A2: a genuine cell cycle regulator?

Abstract Cyclin A2 belongs to the core cell cycle regulators and participates in the control of both S phase and mitosis. However, several observations suggest that it is also endowed with other functions, and our recent data shed light on its involvement in cytoskeleton dynamic and cell motility. From the transcription of its gene to its posttranslational modifications, cyclin A2 regulation reveals the complexity of the regulatory network shaping cell cycle progression. We summarize our current knowledge on this cell cycle regulator and discuss recent findings raising the possibility that cyclin A2 might play a much broader role in epithelial tissues homeostasis.

Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements.

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved.