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), Ca2+-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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
Abstract
Glypican-1 (GPC-1) is a cell surface heparan sulfate proteoglycan that mediates cell proliferation, differentiation and migration. It also possess oncogenic functions in breast, pancreatic ...and lung cancer cells, and is a proposed biomarker for prostate cancer. Despite these data the function of GPC-1 in prostate cancer is not fully known. We addressed this gap-in-knowledge using in vitro and in vivo mouse models. We first showed that GPC-1 was overexpressed in bone metastatic prostate cancer cells (PC-3) but was not detected in lymph node metastatic cancer cells (LNCaP) and non-cancerous prostate cells (RWPE-1). Next, we inhibited GPC-1 expression using shRNA in bone metastatic cancer cells (PC-3), and verified inhibition using western blot analysis and qRT-PCR. Crystal violet staining, trans-well assays, matrigel colony cultures and adhesion assays were used to determine the effect of GPC-1 inhibition on cancer cell growth, migration, spheroid formation and cell adhesion. Inhibition of GPC-1 reduced prostate cancer cell growth, migration and spheroid formation; however, there were no changes in cell adhesion. These in vitro data suggested that GPC-1 played a role in prostate cancer progression by acting as an oncogene. We further investigated this possibility in vivo using athymic (NCr) mice xenografts. Interestingly, inhibition of GPC-1 in PC-3 cells appeared to increase tumor size as compared to scrambled controls. Further analysis showed that that inhibition of GPC-1 increased the tumor expression of MMP-9 and N-cadherin, but not E-cadherin. We hypothesized that the discrepancy between the in vitro and in vivo results may be mediated by interaction of PC-3 cells with the tumor microenvironment. We addressed this hypothesis by testing the effect of tumor conditioned media (TCM) from control and GPC-1 knockdown cells on human bone marrow derived mesenchymal stem cells (hMSC) and fibroblast (HS-27). Treatment of both hMSC and HS-27 cells with TCM from GPC-1 knockdown PC-3 cells increased MMP-9 and interestingly MMP-2 expression and activity as compared to control cells. We also detected increases in the expression of N-Cadherin in a co-culture model. Collectively, these data suggest that GPC-1 acts as an oncogene to enhance prostate cancer cell growth in vitro; however, these data also suggest the GPC-1 may act as a tumor suppressor in vivo. This discrepancy may be due to the interaction off GPC-1 with the tumor microenvironment, which would not be detected in single-cell culture models.
Citation Format: Nhat D. Quach, Matthew Eggert, Deepraj Ghosh, Michelle R. Dawson, Robert Arnold, Brian S. Cummings. Glypican-1: A tumor suppressor or an oncogene in human bone metastatic prostate cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2017-4465
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 A
(sPLA
) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA
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 sPLA
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 sPLA
. 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 sPLA
. sPLA
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 sPLA
.
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