Elevated Expression of Stromal Palladin Predicts Poor Clinical Outcome in Renal Cell Carcinoma

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study

Novel roles for class II Phosphoinositide 3-Kinase C2 beta in signalling pathways involved in prostate cancer cell invasion

Phosphoinositide 3-kinases (PI3Ks) regulate several cellular functions such as proliferation, growth, survival and migration. The eight PI3K isoforms are grouped into three classes and the three enzymes belonging to the class II subfamily (PI3K-C2a, ß and ?) are the least investigated amongst all PI3Ks. Interest on these isoforms has been recently fuelled by the identification of specific physiological roles for class II PI3Ks and by accumulating evidence indicating their involvement in human diseases. While it is now established that these isoforms can regulate distinct cellular functions compared to other PI3Ks, there is still a limited understanding of the signalling pathways that can be specifically regulated by class II PI3Ks. Here we show that PI3K-C2ß regulates mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) activation in prostate cancer (PCa) cells. We further demonstrate that MEK/ERK and PI3K-C2ß are required for PCa cell invasion but not proliferation. In addition we show that PI3K-C2ß but not MEK/ERK regulates PCa cell migration as well as expression of the transcription factor Slug. These data identify novel signalling pathways specifically regulated by PI3K-C2ß and they further identify this enzyme as a key regulator of PCa cell migration and invasion

Differential Expressions of Adhesive Molecules and Proteases Define Mechanisms of Ovarian Tumor Cell Matrix Penetration/Invasion

Epithelial ovarian cancer is an aggressive and deadly disease and understanding its invasion mechanisms is critical for its treatment. We sought to study the penetration/invasion of ovarian tumor cells into extracellular matrices (ECMs) using a fibroblast-derived three-dimensional (3D) culture model and time-lapse and confocal imaging. Twelve ovarian tumor cells were evaluated and classified into distinct groups based on their ECM remodeling phenotypes; those that degraded the ECM (represented by OVCAR5 cells) and those that did not (represented by OVCAR10 cells). Cells exhibiting a distinct ECM modifying behavior were also segregated by epithelial- or mesenchymal-like phenotypes and uPA or MMP-2/MMP-9 expression. The cells, which presented epithelial-like phenotypes, penetrated the ECM using proteases and maintained intact cell-cell interactions, while cells exhibiting mesenchymal phenotypes modified the matrices via Rho-associated serine/threonine kinase (ROCK) in the absence of apparent cell-cell interactions. Overall, this study demonstrates that different mechanisms of modifying matrices by ovarian tumor cells may reflect heterogeneity among tumors and emphasize the need to systematically assess these mechanisms to better design effective therapies

Cancer Cell Invasion Is Enhanced by Applied Mechanical Stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor

Enasidenib induces acute myeloid leukemia cell differentiation to promote clinical response

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies

Enasidenib induces acute myeloid leukemia cell differentiation to promote clinical response

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies

Clonal heterogeneity of acute Myeloid Leukemia treated with the IDH2 inhibitor Enasidenib

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse