Nina Fenouille

Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress, cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62), but its role in the regulation of autophagy is controversial. Here, we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK, thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly, p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV, and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy.

The epithelial-mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion.

During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAFV600E using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAFV600E. Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.

SYK Is a Critical Regulator of FLT3 in Acute Myeloid Leukemia

Cooperative dependencies between mutant oncoproteins and wild-type proteins are critical in cancer pathogenesis and therapy resistance. Although spleen tyrosine kinase (SYK) has been implicated in hematologic malignancies, it is rarely mutated. We used kinase activity profiling to identify collaborators of SYK in acute myeloid leukemia (AML) and determined that FMS-like tyrosine kinase 3 (FLT3) is transactivated by SYK via direct binding. Highly activated SYK is predominantly found in FLT3-ITD positive AML and cooperates with FLT3-ITD to activate MYC transcriptional programs. FLT3-ITD AML cells are more vulnerable to SYK suppression than FLT3 wild-type counterparts. In a FLT3-ITD in vivo model, SYK is indispensable for myeloproliferative disease (MPD) development, and SYK overexpression promotes overt transformation to AML and resistance to FLT3-ITD-targeted therapy.

Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest

Loss of tumor-suppressive pathways that control cellular senescence is a crucial step in malignant transformation. Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that has been recently implicated in tumor suppression of melanoma, a deadly skin cancer derived from pigment-producing melanocytes. However, the mechanism by which Syk suppresses melanoma growth remains unclear. Here, we report that reexpression of Syk in melanoma cells induces a p53-dependent expression of the cyclin-dependent kinase (cdk) inhibitor p21 and a senescence program. We first observed that Syk expression is lost in a subset of melanoma cell lines, primarily by DNA methylation-mediated gene silencing and restored after treatment with the demethylating agent 5-aza-2-deoxycytidine. We analyzed the significance of epigenetic inactivation of Syk and found that reintroduction of Syk in melanoma cells dramatically reduces clonogenic survival and three-dimensional tumor spheroid growth and invasion. Remarkably, melanoma cells reexpressing Syk display hallmarks of senescent cells, including reduction of proliferative activity and DNA synthesis, large and flattened morphology, senescence-associated beta-galactosidase activity, and heterochromatic foci. This phenotype is accompanied by hypophosphorylated retinoblastoma protein (Rb) and accumulation of p21, which depends on functional p53. Our results highlight a new role for Syk tyrosine kinase in regulating cellular senescence and identify Syk-mediated senescence as a novel tumor suppressor pathway the inactivation of which may contribute to melanoma tumorigenicity.

Persistent Activation of the Fyn/ERK Kinase Signaling Axis Mediates Imatinib Resistance in Chronic Myelogenous Leukemia Cells through Upregulation of Intracellular SPARC

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization, growth factor availability, cell adhesion, differentiation, and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study, we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity, whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably, we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly, SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings, increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together, our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC.

Tumour-derived SPARC drives vascular permeability and extravasation through endothelial VCAM1 signalling to promote metastasis

Disruption of the endothelial barrier by tumour-derived secreted factors is a critical step in cancer cell extravasation and metastasis. Here, by comparative proteomic analysis of melanoma secretomes, we identify the matricellular protein SPARC as a novel tumour-derived vascular permeability factor. SPARC deficiency abrogates tumour-initiated permeability of lung capillaries and prevents extravasation, whereas SPARC overexpression enhances vascular leakiness, extravasation and lung metastasis. SPARC-induced paracellular permeability is dependent on the endothelial VCAM1 receptor and p38 MAPK signalling. Blocking VCAM1 impedes melanoma-induced endothelial permeability and extravasation. The clinical relevance of our findings is highlighted by high levels of SPARC detected in tumour from human pulmonary melanoma lesions. Our study establishes tumour-produced SPARC and VCAM1 as regulators of cancer extravasation, revealing a novel targetable interaction for prevention of metastasis.

The p53/p21Cip1/ Waf1 pathway mediates the effects of SPARC on melanoma cell cycle progression

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, belongs to the family of matricellular proteins that modulate cell–matrix interactions and cellular functions. SPARC is highly expressed in melanoma, and we reported that SPARC promotes epithelial/mesenchymal‐like changes and cell migration. Here, we used siRNA and conditional shRNA to investigate the contribution of tumor‐derived SPARC to melanoma cell growth in vitro and in vivo. We found that depletion of SPARC induces G2/M cell cycle arrest and tumor growth inhibition with activation of p53 and induction of p21Cip1/Waf1 acting as a checkpoint, preventing efficient mitotic progression. In addition, we demonstrate that reduced mesenchymal features and the invasive potential of SPARC‐silenced cells are independent of p21Cip1/Waf1 induction and cell cycle arrest. Importantly, overexpression of SPARC reduces p53 protein levels and leads to an increase in cell number during exponential growth. Our findings indicate that in addition to its well‐known function as a mediator of melanoma cell migration and tumor–host interactions, SPARC regulates, in a cell‐autonomous manner, cell cycle progression and proliferation through the p53/p21Cip1/Waf1 pathway.

Imatinib triggers mesenchymal-like conversion of CML cells associated with increased aggressiveness

Chronic myelogenous leukemia (CML) is a cytogenetic disorder resulting from the expression of p210BCR-ABL. Imatinib, an inhibitor of BCR-ABL, has emerged as the leading compound to treat CML patients. Despite encouraging clinical results, resistance to imatinib represents a major drawback for therapy, as a substantial proportion of patients are refractory to this treatment. Recent publications have described the existence of a small cancer cell population with the potential to exhibit the phenotypic switch responsible for chemoresistance. To investigate the existence of such a chemoresistant cellular subpopulation in CML, we used a two-step approach of pulse and continuous selection by imatinib in different CML cell lines that allowed the emergence of a subpopulation of adherent cells (IM-R Adh) displaying an epithelial-mesenchymal transition (EMT)-like phenotype. Overexpression of several EMT markers was observed in this CML subpopulation, as well as in CD34(+) CML primary cells from patients who responded poorly to imatinib treatment. In response to imatinib, this CD44(high)/CD24(low) IM-R Adh subpopulation exhibited increased adhesion, transmigration and invasion in vitro and in vivo through specific overexpression of the αVβ3 receptor. FAK/Akt pathway activation following integrin β3 (ITGβ3) engagement mediated the migration and invasion of IM-R Adh cells, whereas persistent activation of ERK counteracted BCR-ABL inhibition by imatinib, promoting cell adhesion-mediated resistance.

Calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance in colorectal cancer xenografts†

CPT‐11 (irinotecan), the first‐line chemotherapy for advanced stage colorectal cancer, remains inactive in about half of patients (primary chemoresistance) and almost all initial responders develop secondary resistance after several courses of treatment (8 months on average). Nude mice bearing HT‐29 colon cancer xenografts were treated with CPT‐11 and/or an NF‐κB inhibitor for two courses. We confirm that NF‐κB inhibition potentiated CPT‐11 anti‐tumoural effect after the first course of treatment. However, tumours grew again at the end of the second course of treatment, generating resistant tumours. We observed an increase in the basal NF‐κB activation in resistant tumours and in two resistant sublines, either obtained from resistant HT‐29 tumours (HT‐29R cells) or generated in vitro (RSN cells). The decrease of NF‐κB activation in HT‐29R and RSN cells by stable transfections with the super‐repressor form of IκBα augmented their sensitivity to CPT‐11. Comparing gene expression profiles of HT‐29 and HT‐29R cells, we identified the S100A10/Annexin A2 complex and calpain 2 as over‐expressed potential NF‐κB inducers. SiRNA silencing of calpain 2 but not of S100A10 and/or annexin A2, resulted in a decrease in NF‐κB activation, an increase in cellular levels of IκBα and a partial restoration of the CPT‐11 sensitivity in both HT‐29R and RSN cells, suggesting that calpain 2‐dependent IκBα degradation mediates CPT‐11 secondary resistance. Thus, targeted therapies directed against calpain 2 may represent a novel strategy to enhance the anti‐cancer efficacy of CPT‐11. Copyright

Mechanism of action of the multikinase inhibitor Foretinib

Mitotic catastrophe (MC) is induced when stressed cells enter prematurely or inappropriately into mitosis and can be caused by ionizing radiation and anticancer drugs. Foretinib is a multikinase inhibitor whose mechanism of action is incompletely understood. We investigated here the effect of Foretinib on chronic myelogenous leukemia (CML) cell lines either sensitive (IM-S) or resistant (IM-R) to the tyrosine kinase inhibitor Imatinib. Foretinib decreased viability and clonogenic potential of IM-S and IM-R CML cells as well. Foretinib-treated cells exhibited increased size, spindle assembly checkpoint anomalies and enhanced ploidy that collectively evoked mitotic catastrophe (MC). Accordingly, Foretinib-stimulated CML cells displayed decreased expression of Cdk1, Cyclin B1 and Plk1. In addition, Foretinib triggered caspase-2 activation that precedes mitochondrial membrane permeabilization. Accordingly, z-VAD-fmk and a caspase-2 siRNA abolished Foretinib-mediated cell death but failed to affect MC, indicating that Foretinib-mediated apoptosis and MC are two independent events. Anisomycin, a JNK activator, impaired Foretinib-induced MC and inhibition or knockdown of JNK phenotyped its effect on MC. Moreover, we found that Foretinib acted as a potent inhibitor of JNK. Importantly, Foretinib exhibited no or very little effect on normal peripheral blood mononuclear cells, monocytes or melanocytes cells but efficiently inhibited the clonogenic potential of CD34+ cell from CML patients. Collectively, our data show that the multikinase inhibitor Foretinib induces MC in CML cells and other cell lines via JNK-dependent inhibition of Plk1 expression and triggered apoptosis by a caspase 2-mediated mechanism. This unusual mechanism of action may have important implications for the treatment of cancer.