Senescent mesenchymal stem cells remodel extracellular matrix driving breast cancer cells to a more-invasive phenotype.

Mesenchymal stem cells (MSCs) are essential for the regenerative process; however, biological aging and environmental stress can induce senescence - an irreversible state of growth arrest - that not only affects the behavior of cells but also disrupts their ability to restore tissue integrity. While abnormal tissue properties, including increased extracellular matrix stiffness, are linked with the risk of developing breast cancer, the role and contribution of senescent MSCs to the disease progression to malignancy are not well understood. Here, we investigated senescence-associated biophysical changes in MSCs and how this influences cancer cell behavior in a 3D matrix interface model. Although senescent MSCs were far less motile than pre-senescent MSCs, they induced an invasive breast cancer phenotype, characterized by increased spheroid growth and cell invasion in collagen gels. Further analysis of collagen gels using second-harmonic generation showed increased collagen density when senescent MSCs were present, suggesting that senescent MSCs actively remodel the surrounding matrix. This study provides direct evidence of the pro-malignant effects of senescent MSCs in tumors.

Liposomes Targeting P21 Activated Kinase-1 (PAK-1) and Selective for Secretory Phospholipase A2 (sPLA2) Decrease Cell Viability and Induce Apoptosis in Metastatic Triple-Negative Breast Cancer Cells.

P21 activated kinases (or group I PAKs) are serine/threonine kinases whose expression is altered in prostate and breast cancers. PAK-1 activity is inhibited by the small molecule "Inhibitor targeting PAK-1 activation-3" (IPA-3), which has selectivity for PAK-1 but is metabolically unstable. Secretory Group IIA phospholipase A2 (sPLA2) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA2, called Secretory Phospholipase Responsive liposomes or SPRL-IPA-3, and demonstrated their ability to alter prostate cancer growth. The efficacy of SPRL against other types of cancers is not well understood. We addressed this limitation by determining the ability of SPRL to induce cell death in a diverse panel of cells representing different stages of breast cancer, including the invasive but non-metastatic MCF-7 cells, and metastatic triple-negative breast cancer (TNBC) cells such as MDA-MB-231, MDA-MB-468, and MDA-MB-435. We investigated the role of sPLA2 in the disposition of these liposomes by comparing the efficacy of SPRL-IPA-3 to IPA-3 encapsulated in sterically stabilized liposomes (SSL-IPA-3), a formulation shown to be less sensitive to sPLA2. Both SSL-IPA-3 and SPRL-IPA-3 induced time- and dose-dependent decreases in MTT staining in all cell lines tested, but SPRL-IPA-3-induced effects in metastatic TNBC cell lines were superior over SSL-IPA-3. The reduction in MTT staining induced by SPRL-IPA-3 correlated to the expression of Group IIA sPLA2. sPLA2 expression also correlated to increased induction of apoptosis in TNBC cell lines by SPRL-IPA-3. These data suggest that SPRL-IPA-3 is selective for metastatic TNBC cells and that the efficacy of SPRL-IPA-3 is mediated, in part, by the expression of Group IIA sPLA2.

Spatially coordinated changes in intracellular rheology and extracellular force exertion during mesenchymal stem cell differentiation.

The mechanical properties within the cell are regulated by the organization of the actin cytoskeleton, which is linked to the extracellular environment through focal adhesion proteins that transmit force. Chemical and mechanical stimuli alter the organization of cytoskeletal actin, which results in changes in cell shape, adhesion, and differentiation. By combining particle-tracking microrheology and traction force cytometry, we can monitor the mechanical properties of the actin meshwork and determine how changes in the intracellular network contribute to force generation. In this study, we investigated the effects of chemical (differentiation factors) and mechanical (substrate rigidity) stimuli important in mesenchymal stem cell (MSC) differentiation on the intracellular mechanics and traction stress generation. We found the presence of adipogenic factors resulted in stiffening of the actin meshwork regardless of substrate rigidity. In contrast, these factors increased traction stresses on hard substrates, which was associated with increased expression of contractility genes. Furthermore, MSCs cultured on hard substrates expressed both adipogenic and osteogenic markers indicative of mixed differentiation. On hard substrates, heterogeneity in the local elastic modulus-traction stress correlation was also increased in response to adipogenic factors, indicating that these mechanical properties may be reflective of differences in the level of MSC differentiation. These results suggest intracellular rheology and traction stress generation are spatially regulated and contribute insight into how single cell mechanical forces contribute to MSC differentiation.

Ovarian Cancer Exosomes Trigger Differential Biophysical Response in Tumor-Derived Fibroblasts.

Exosomes are cell-secreted microvesicles that play important roles in epithelial ovarian cancer (EOC) progression, as they are constantly secreted into ascites fluids. While cells spontaneously release exosomes, alterations in intracellular calcium or extracellular pH can release additional exosomes. Yet, little is known about how these exosomes compare to those that are continuously released without stimulation and how they mediate cellular activities important in cancer progression. Here, we demonstrate that chelation of extracellular calcium leads to release of chelation-induced exosomes (CI-exosomes) from OVCAR-3 EOC cells. CI-exosomes display a unique miRNA profile compared to naturally secreted exosomes (SEC-exosomes). Furthermore, treatment with CI- and SEC-exosomes leads to differential biophysical and functional changes including, adhesion and migration in EOC-derived fibroblasts that suggest the development of a malignant tumor microenvironment. This result highlights how tumor environmental factors contribute to heterogeneity in exosome populations and how different exosome populations mediate diversity in stromal cell behavior.

Effect of P21-activated kinase 1 (PAK-1) inhibition on cancer cell growth, migration, and invasion.

P21-activated kinase-1 (PAK-1) is a serine/threonine kinase involved in multiple signaling pathways that mediate cellular functions such as cytoskeletal motility, cell proliferation, and survival. PAK-1 expression is altered in various cancers, including prostate and breast. Our recent studies showed that prostate cancer cells expressing higher levels of PAK-1 were resistant to the cytotoxic effects of the PAK-1 inhibitor, inhibitor targeting PAK-1 activation-3 (IPA-3), compared to those with lower expression. This study expanded these findings to other cancers (breast and melanoma) by testing the hypothesis that genetic and pharmacological inhibition of PAK-1 alters cell growth, migration, and invasion in prostate, breast, and skin cancer cell lines. We also tested the specificity of IPA-3 for PAK-1 and the hypothesis that gene silencing of PAK-1 altered the efficacy of sterically stabilized liposomes (SSL) containing IPA-3 (SSL-IPA-3). PAK-1 expression was identified in four different breast cancer cell lines, and in a melanoma cell line. The expression of PAK-1 correlated to the IC50 of IPA-3 as measured by MTT staining. PAK-1 inhibition using shRNA correlated with decreased cell migration and invasion in prostate cancer DU-145 and breast cancer MCF-7 cells. Decreased migration and invasion also correlated to decreased expression of E-cadherin and alterations in C-X-C Chemokine Receptor type 4 and Homing Cell Adhesion Molecule expression. PAK-1 inhibition increased the cytotoxicity of IPA-3, and the cytotoxicity of SSL-IPA-3 to levels comparable to that of free drug. These data demonstrate that both pharmacological and molecular inhibition of PAK-1 decreased growth in prostate, breast, and melanoma cancer cell lines, and increased the toxicity of IPA-3 and its liposomal formulation. These data also show the specificity of IPA-3 for PAK-1, are some of the first data suggesting that IPA-3 is a therapeutic treatment for breast cancer and melanoma, and demonstrate the efficacy of liposome-encapsulated IPA-3 in breast cancer cells.

Paradoxical Role of Glypican-1 in Prostate Cancer Cell and Tumor Growth.

Recent studies suggest that glypican-1 (GPC-1) is a biomarker for prostate cancer, but there are few studies elucidating the role of GPC-1 in prostate cancer progression. We observed high expression of GPC-1 in more aggressive prostate cancer cell lines such as PC-3 and DU-145. While inhibition of GPC-1 expression in PC-3 cells decreased cell growth and migration in vitro, it surprisingly increased cell proliferation and migration in DU-145 cells, suggesting that the role of GPC-1 is cell type-dependent. Further, GPC-1 inhibition increased PC-3 tumor size in NCr nude mice xenografts. We hypothesized that the discrepancy between the in vitro and in vivo data is mediated by stromal cells in the tumor microenvironment. Thus, we tested the effect of tumor conditioned media (TCM) on gene expression in human mesenchymal stem cells and fibroblasts. Treatment of stromal cells with TCM from PC-3 cells transfected with GPC-1 shRNA increased the expression of migration markers, endocrine/paracrine biomolecules, and extracellular matrix components. Additionally, the decreased cell growth in GPC-1 knockdown PC-3 cells was rescued by coculturing with stromal cells. These data demonstrate the paradoxical role that GPC-1 plays in prostate cancer cell growth by interacting with stromal cells and through ECM remodeling and endocrine/paracrine signaling.

Secretory phospholipase A2 enzymes as pharmacological targets for treatment of disease.

Phospholipase A2 (PLA2) cleave phospholipids preferentially at the sn-2 position, liberating free fatty acids and lysophospholipids. They are classified into six main groups based on size, location, function, substrate specificity and calcium requirement. These classes include secretory PLA2 (sPLA2), cytosolic (cPLA2), Ca(2+)-independent (iPLA2), platelet activating factor acetylhydrolases (PAF-AH), lysosomal PLA2 (LyPLA2) and adipose specific PLA2 (AdPLA2). It is hypothesized that PLA2 can serve as pharmacological targets for the therapeutic treatment of several diseases, including cardiovascular diseases, atherosclerosis, immune disorders and cancer. Special emphasis has been placed on inhibitors of sPLA2 isoforms as pharmacological moieties, mostly due to the fact that these enzymes are activated during inflammatory events and because their expression is increased in several diseases. This review focuses on understanding how sPLA2 isoform expression is altered during disease progression and the possible therapeutic interventions to specifically target sPLA2 isoforms, including new approaches using nano-particulate-based strategies.

Convergent regulation of neuronal differentiation and Erk and Akt kinases in human neural progenitor cells by lysophosphatidic acid, sphingosine 1-phosphate, and LIF: specific roles for the LPA1 receptor.

The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.

Differential mechanical response of mesenchymal stem cells and fibroblasts to tumor-secreted soluble factors.

The progression of neoplastic malignancies is a complex process resulting not only from the accumulation of mutations within tumor cells, but also modulation of the tumor microenvironment. Recent advances have shown that the recruitment and subsequent heterotypic interactions of stromal cells--including fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs)--are crucial for carcinogenesis. Though extensive work has been done analyzing the signals that recruit these cells, the governing mechanical properties have not been fully investigated. Here, we report that despite their initial similarities, MSCs respond not only faster but also more dramatically to pro-migratory tumor-secreted soluble factors. Utilizing multiple particle tracking microrheology to probe the cytoskeletal mechanical properties, we show that MSCs stiffen completely within one hour, three times faster than fibroblasts. In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion. Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1. These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs.