WISP1 Research Articles

Circulating WNT1 inducible signaling pathway protein 1 (WISP1) is a novel marker of visceral obesity

Circulating WNT1 inducible signaling pathway protein 1 (WISP1) is a novel marker of visceral obesity

WNT-1 inducible signaling pathway protein-1 enhances growth and tumorigenesis in human breast cancer.

WNT1 inducible signaling pathway protein 1 (WISP1) plays a key role in many cellular functions in a highly tissue-specific manner; however the role of WISP1 in breast cancer is still poorly understood. Here, we demonstrate that WISP1 acts as an oncogene in human breast cancer. We demonstrated that human breast cancer tissues had higher WISP1 mRNA expression than normal breast tissues and that treatment of recombinant WISP1 enhanced breast cancer cell proliferation. Further, ectopic expression of WISP1 increased the growth of breast cancer cells in vitro and in vivo. WISP1 transfection also induced epithelial-mesenchymal-transition (EMT) in MCF-7 cells, leading to higher migration and invasion. During this EMT-inducing process, E-cadherin was repressed and N-cadherin, snail, and β-catenin were upregulated. Filamentous actin (F-actin) remodeling and polarization were also observed after WISP1 transfection into MCF-7 cells. Moreover, forced overexpression of WISP1 blocked the expression of NDRG1, a breast cancer tumor suppressor gene. Our study provides novel evidence that WISP1-modulated NDRG1 gene expression is dependent on a DNA fragment (−128 to +46) located within the human NDRG1 promoter. Thus, we concluded that WISP1 is a human breast cancer oncogene and is a potential therapeutic target.

IRE1 inhibition affects the expression of insulin-like growth factor binding protein genes and modifies its sensitivity to glucose deprivation in U87 glioma cells.

The aim of the present study was to investigate the effect of inhibition of endoplasmic reticulum stress signaling mediated by IRE1/ERN1 (inositol-requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) on the expression of genes encoding different groups of insulin-like growth binding proteins (IGFBP6 and IGFBP7) and CCN family (IGFBP8/CTGF/CCN2, IGFBP9/NOV/CCN3, IGFBP10/CYR61/CCN1, WISP1/CCN4, and WISP2/CCN5) and its sensitivity to glucose deprivation in U87 glioma cells. The expression of IGFBP6, IGFBP7, IGFBP8, IGFBP9, IGFBP10, WISP1, and WISP2 genes was studied by qPCR in control U87 glioma cells (wild-type) and its subline with IRE1 signaling enzyme loss of function upon glucose deprivation. The expression of IGFBP8, IGFBP9, and WISP2 genes was up-regulated in control glioma cells upon glucose deprivation with most significant changes for IGFBP9 gene. At the same time, the expression of IGFBP6, IGFBP10, and WISP1 genes was resistant to glucose deprivation in these glioma cells, but the IGFBP7 gene expression was down-regulated. The inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells modified the sensitivity of most studied gene expressions to glucose deprivation condition: introduced sensitivity of IGFBP10 and WISP1 genes to glucose deprivation, enhanced the effect of this deprivation on IGFBP7 and IGFBP9 gene expressions, and reduced this effect on WISP2 gene and induced suppressive effect of glucose deprivation on the expression of IGFBP8 gene. Furthermore, the inhibition of IRE1 strongly affected the expression of all studied genes in glioma cells upon regular growing condition in gene specific manner: up-regulated the expression levels of IGFBP7, IGFBP8, IGFBP10, WISP1, and WISP2 genes and down-regulated the IGFBP6 and IGFBP9 genes. The data of this investigation demonstrate that the expression of IGFBP7, IGFBP8, IGFBP9, and WISP2 genes are sensitive to glucose deprivation in U87 glioma cells and that inhibition of IRE1 signaling enzyme function may significantly affect the expression of all studied genes in the presence of glucose as well as modify the effect of glucose deprivation on the expression of most studied genes. These data also show that proteins encoded by these genes may participate in the regulation of metabolic and proliferative processes via IGF/INS receptors and possibly other signaling pathways as well, via IRE1 signaling, which is acentral mediator of the unfolded protein response and an important component of the tumor growth and metabolic diseases.

New Insights for Oxidative Stress and Diabetes Mellitus.

The release of reactive oxygen species (ROS) and the generation of oxidative stress are considered critical factors for the pathogenesis of diabetes mellitus (DM), a disorder that is growing in prevalence and results in significant economic loss. New therapeutic directions that address the detrimental effects of oxidative stress may be especially warranted to develop effective care for the millions of individuals that currently suffer from DM. The mechanistic target of rapamycin (mTOR), silent mating type information regulation 2 homolog 1 (S. cerevisiae) (SIRT1), and Wnt1 inducible signaling pathway protein 1 (WISP1) are especially justified to be considered treatment targets for DM since these pathways can address the complex relationship between stem cells, trophic factors, impaired glucose tolerance, programmed cell death pathways of apoptosis and autophagy, tissue remodeling, cellular energy homeostasis, and vascular biology that greatly impact the biology and disease progression of DM. The translation and development of these pathways into viable therapies will require detailed understanding of their proliferative nature to maximize clinical efficacy and limit adverse effects that have the potential to lead to unintended consequences.

Green tea polyphenol EGCG reverse cisplatin resistance of A549/DDP cell line through candidate genes demethylation

Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been extensively studied as a potential demethylating agent. Our hypothesis is that EGCG could resensitize non-small-cell lung cancer (NSCLC) cells to cisplatin (DDP) through candidate genes demethylation. The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. MTT, colony formation assay, flow cytometric analysis, Hoechst staining, real time-PCR, quantitative methylation-specific PCR and in vivo experiments were performed in this study. EGCG+DDP treatment significantly caused proliferation inhibition, cell cycle arrest in G1 phase, increase of apoptosis in A549/DDP cells, along with inhibition of DNA methyltransferase (DNMT) activity and histone deacetylase (HDAC) activity, reversal of hypermethylated status and downregulated expression of GAS1, TIMP4, ICAM1 and WISP2 gene in A549/DDP cells. Furthermore, pre-treatment with EGCG followed by DDP caused significant tumor inhibition in vivo. Methylation levels of GAS1, TIMP4, ICAM1 and WISP2 were decreased and their expression levels were increased in EGCG-treatment groups, but only combinatorial treatment group caused growth inhibition. In conclusion, we identified EGCG pretreatment resensitized cells to DDP, along with the demethylation and restoration of expression of candidate genes.

Emerging Roles of Wisp-1 and SFRP4 in Proliferation and Differentiation Potential of Wharton’s Jelly Mesenchymal Stem/Stromal Cells

We have previously shown (Batsali A et al., Blood 2013:122, 1212) that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton’s jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. Provided that the WNT-pathways are involved in proliferation and differentiation of BM-MSCs, we assumed that the aforementioned findings might be attributed, at least in part, to aberrant WNT-signaling in WJ-MSCs. In support of this hypothesis, we found that gene expression of the Wnt antagonist sFRP4, a promoter of adipogenesis in human adipose tissue-derived MSCs, was significantly down-regulated in WJ-MSCs and that mRNA levels of WNT-induced secreted protein-1, (WISP-1), a regulator of osteogenesis in BM-MSCs, were also significantly reduced in WJ-MSCs. These observations imply a connection between these WNT-associated molecules and the biological properties of WJ-MSCs, which requires, however, further investigation.The present study was undertaken so as to explore the effects of WISP-1 and sFRP4 in growth and differentiation of ex-vivo expanded WJ-MSCs.MSCs were isolated from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=5) after written informed consent of the family. MSCs were in vitro expanded, re-seeded for a total of 4 passages (P) and phenotypically characterized by flow cytometry at P3. WJ-MSCs were grown in the absence or presence of rhWISP-1 or rhsFRP4 and cell proliferation was assessed by a methyl-triazolyl-tetrazolium (MTT)-assay. In addition, WJ-MSCs were induced to differentiate in vitro to osteoblasts and adipocytes, in the absence or presence of rhWISP-1 or rhsFRP4 respectively. Differentiation was quantified by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes by real time RT-PCR. Relative gene expression was calculated by the ΔCt method. The expression of WISP-1 and sFRP4 by non-differentiated WJ- and BM-MSCs as well as by WJ-MSCs during osteogenesis and adipogenesis, respectively, was also evaluated by real time RT-PCR.Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. In line with our previous work WISP-1 and sFRP4 mRNA were significantly decreased in WJ-MSCs, compared to BM-MSCs.To explore the role of WISP-1 in WJ-MSCs’ growth we cultured cells in the presence of 50 ng/ml or 100 ng/ml rhWISP1 and assessed cell proliferation at multiple time points, throughout a 14-day culture. WISP-1 treatment did not lead to any significant effect in cell numbers. Next, we investigated the time course of WISP1 gene expression during osteoinduction. In all samples, WISP1 mRNA levels increased during osteogenesis. As compared to day0 (exposure to osteogenic medium), the increase in gene expression reached statistical significance at days 7 and 14. Furthermore, WISP-1 gene expression was significantly higher at day 14, compared to day 7. To investigate the functional effects of WISP1 on the osteoblastic differentiation of WJ-MSCs, cells were cultured for 7 days in osteogenic medium supplemented with 50ng/ml rh-WISP1. A significant increase in the expression of RUNX2 and ALP was detected, compared to non-treated cells.To investigate the impact of sFRP4 in WJ-MSC’s proliferation we exposed cells to 20nM rhsFRP4 for 14 days. Live cell numbers, at various time points, were significantly reduced in treated cells. Regarding the time course of sFRP4 expression during adipogenic differentiation, sFRP4 mRNA levels increased during adipogenesis reaching statistical significance at days7 and 14, as compared to day0. In addition, sFRP4 gene expression was significantly higher at day 14 as compared to day 7. Finally, when cells underwent adipogenic differentiation in the presence of rhSFRP4, a significant increase in PPARG and CEBPA mRNA levels was detected at day 14, as compared to non-treated cellsCollectively, our results suggest that WISP-1 and sFRP4 may be actively implicated in proliferation and differentiation of WJ-MSCs. The functional role of these WNT-related molecules in the biology of WJ-MSCs requires deeper understanding, in view of the growing interest for the use of WJ-MSCs in cell-based therapies. DisclosuresNo relevant conflicts of interest to declare.

WISP1 polymorphisms contribute to platinum-based chemotherapy toxicity in lung cancer patients.

Platinum-based chemotherapy toxicity is always one of the serious problems from which lung cancer patients suffer. The genetic polymorphism of WISP1 was revealed to be associated with susceptibility and platinum-based chemotherapy response in our previous studies. In this study, we aimed to investigate the relationship of WISP1 genetic polymorphisms with platinum-based chemotherapy toxicity in lung cancer patients. A total of 412 lung cancer patients were enrolled in this study, and 28 polymorphisms of the WISP1 gene were genotyped by SequenomMassARRAY. We found that WISP1 polymorphisms (rs2929965, rs2929969, rs2929970, rs2929973 and rs754958) were related to the overall chemotherapy toxicity of lung cancer in subgroup analyses. Rs16904853, rs2929970, rs2977549 and rs2977551 (p = 0.021, 0.028, 0.024, 0.048, respectively) polymorphisms were significantly associated with hematologic toxicity. Rs2929946, rs2929970, rs2977519, rs2977536, rs3739262 and rs754958 (p = 0.031, 0.046, 0.029, 0.016, 0.042, 0.035, respectively) polymorphisms were significantly associated with the gastrointestinal toxicity of lung cancer. Genotypes of WISP1 may be novel and useful biomarkers for predicting platinum-based chemotherapy toxicity in lung cancer patients.

WISP1: Clinical insights for a proliferative and restorative member of the CCN family.

As a proliferative and restorative entity, Wnt1 inducible signaling pathway protein 1 (WISP1) is emerging as a novel target for a number of therapeutic strategies that are relevant for disorders such as traumatic injury, neurodegeneration, musculoskeletal disorders, cardiovascular disease, pulmonary compromise, and control of tumor growth as well as distant metastases. WISP1, a target of the wingless pathway Wnt1, oversees cellular mechanisms that include apoptosis, autophagy, cellular migration, stem cell proliferation, angiogenesis, immune cell modulation, and tumorigenesis. The signal transduction pathways of WISP1 are broad and involve phosphoinositide 3-kinase (PI 3-K), protein kinase B (Akt), mitogen activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK), caspases, forkhead transcription factors, sirtuins, c-myc, glycogen synthase kinase -3β (GSK-3β), β-catenin, miRNAs, and the mechanistic target of rapamaycin (mTOR). Ultimately, these signal transduction pathways of WISP1 can result in varied and sometimes unpredictable outcomes especially for cell survival, tissue repair, and tumorigenesis that demand increased insight into the critical role WISP1 holds for cellular biology and clinical medicine.

MiR-92a regulates TGF-β1-induced WISP1 expression in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of idiopathic interstitial pneumonia. MicroRNAs (miRNAs), short, single-stranded RNAs that regulate protein expression in a post-transcriptional manner, have recently been demonstrated to contribute to IPF pathogenesis. We have previously identified WNT1-inducible signaling pathway protein 1 (WISP1) as a highly expressed pro-fibrotic mediator in IPF, but the underlying mechanisms resulting in increased WISP1 expression, remain elusive. Here, we investigated whether WISP1 is a target of miRNA regulation. We applied a novel supervised machine learning approach, which predicted miR-30a/d and miR-92a target sites in regions of the human WISP1 3′UTR preferentially bound by the miRNA ribonucleoprotein complex. Both miRNAs were decreased in IPF samples, whereas WISP1 protein was increased. We demonstrated further that transforming growth factor (TGF)-β1-induced WISP1 expression in primary lung fibroblasts in vitro and lung homogenates in vivo. Notably, miR-30a and miR-92a reversed TGF-β1-induced WISP1 mRNA expression in lung fibroblasts. Moreover, miR-92a inhibition increased WISP1 protein expression in lung fibroblasts. An inverse relationship for WISP1 and miR-92a was found in a TGF-β1 dependent lung fibrosis model in vivo. Finally, we found significantly increased WISP1 expression in primary IPF fibroblasts, which negatively correlated with miR-92a level ex vivo. Altogether, our findings indicate a regulatory role of miR-92a for WISP1 expression in pulmonary fibrosis.

ABSTRACT 488

Background and aims: Identifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. The WNT signal pathway plays a role in lung development and in asthma pathogenesis.However whether genetic polymorphisms of Wnt signal pathway are associated with lung function in asthma patients remain unclear. Aims: We genotyped two single nucleotide polymorphisms (SNPs, rs2929973 and rs6581612) involved in the Wnt signal pathway in a cohort of 560 Chinese Han child asthmatics. Methods: Associations between each SNP and lung function, in a baseline exam, were tested using multiple linear regression models. Results: We found that rs2929973 of the WISP1 gene was significantly associated with forced expiratory volume in 1 s (FEV1), with the G allele conferring significantly lower FEV1 values. However, the rs6581612 SNP of the WIF1 gene was not associated with differences in FEV1 values. Conclusions: We conclude that genetic variants in Wnt are associated with lung function and suggest that Wnt participates in inflammatory pathways that have an impact on the level of lung function.

Wnt1 inducible signaling pathway protein 1 (WISP1) is a novel adipokine linked to inflammation and insulin resistance in visceral fat

Introduction: WISP1 is a member of the CCN family of extracellular matrix proteins and target gene of Wnt signaling pathway. Here we show that WISP1 is released from adipocytes and WISP1 expression correlate with weight loss and signs of insulin resistance.

Genetic imbalances detected by multiplex ligation-dependent probe amplification in a cohort of patients with oral squamous cell carcinoma—the first step towards clinical personalized medicine

Oral tumors are a growing health problem worldwide; thus, it is mandatory to establish genetic markers in order to improve diagnosis and early detection of tumors, control relapses and, ultimately, delineate individualized therapies. This study was the first to evaluate and discuss the clinical applicability of a multiplex ligation-dependent probe amplification (MLPA) probe panel directed to head and neck cancer. Thirty primary oral squamous cell tumors were analyzed using the P428 MLPA probe panel. We detected genetic imbalances in 26 patients and observed a consistent pattern of distribution of genetic alterations in terms of losses and gains for some chromosomes, particularly for chromosomes 3, 8, and 11. Regarding the latter, some specific genes were highlighted due to frequent losses of genetic material--RARB, FHIT, CSMD1, GATA4, and MTUS1--and others due to gains--MCCC1, MYC, WISP1, PTK2, CCND1, FGF4, FADD, and CTTN. We also verified that the gains of MYC and WISP1 genes seem to suggest higher propensity of tumors localized in the floor of the mouth. This study proved the value of this MLPA probe panel for a first-tier analysis of oral tumors. The probemix was developed to include target regions that have been already shown to be of diagnostic/prognostic relevance for oral tumors. Furthermore, this study emphasized several of those specific genetic targets, suggesting its importance to oral tumor development, to predict patients' outcomes, and also to guide the development of novel molecular therapies.

Effects of Tension Force on Proliferation and Differentiation of Human Periodontal Ligament Cells Induced by Lipopolysaccharides

Human periodontal ligament cells (hPDLCs), with the potential for multi-directional differentiation and reproduction, are the target cells of orthodontic tooth movement. The aim of this study was to examine the effect of mechanical tension force and lipopolysaccharides (LPS) on hPDLCs and whether they induce proliferative and differentiated characters in vitro. Tension force was applied to hPDLCs stimulated with and without LPS for 24 hrs. Real-time polymerase chain reaction (qPCR) was carried out to analyze the mRNA expression of Cyclin 2 (CCND2), WNT1 inducible signaling pathway protein 1 (WISP1), runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP). Analysis of variance (ANOVA) was used for statistical analysis. Significant differences were indicated by P < 0.05. The results showed that tension force promoted the mRNA expression of both the proliferation-related genes (CCND2 and WISP1) and differentiation-related genes (RUNX2 and ALP), and that both were enhanced by the simulation of LPS. In addition, the relative expression ratios CCND2/RUNX2 and CCND2/ALP both increased significantly after the application of tension, and this effect was further enhanced by LPS. All results indicated that with the assessed level of mechanical force loading, tension could promote both the proliferation and differentiation of hPDLCs, which could be enhanced by LPS, and that proliferation is promoted to a greater extent than differentiation. These findings may be valuable for understanding the importance of the application of suitable mechanical force in periodontal remodeling, especially in the process of orthodontic tooth movement with inflammation.

Apoptosis Signal-Regulating Kinase 1 Is Involved in WISP-1–Promoted Cell Motility in Human Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) has a tendency to migrate and metastasize. WNT1-inducible signaling pathway protein 1 (WISP-1) is a cysteine-rich protein that belongs to the Cyr61, CTGF, Nov (CCN) family of matrix cellular proteins. The effect of WISP-1 on human OSCC cells, however, is unknown. Here, we showed that WISP-1 increased cell migration and intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. Pretreatment of cells with integrin αvβ3 monoclonal antibody (mAb) significantly abolished WISP-1–induced cell migration and ICAM-1 expression. On the other hand, WISP-1–mediated cell motility and ICAM-1 upregulation were attenuated by ASK1, JNK, and p38 inhibitor. Furthermore, WISP-1 also enhanced activator protein 1 (AP-1) activation, and the integrin αvβ3 mAb, and ASK1, JNK, and p38 inhibitors reduced WISP-1–mediated AP-1 activation. Moreover, WISP-1 and ICAM-1 expression correlated with the tumor stage of patients with OSCC. Our results indicate that WISP-1 enhances the migration of OSCC cells by increasing ICAM-1 expression through the αvβ3 integrin receptor and the ASK1, JNK/p38, and AP-1 signal transduction pathways.

A role for WNT1-inducible signaling protein-1 in airway remodeling in a rat asthma model

Over-expression of WISP1 has been described in multi-organ fibrosis and tissue remodeling. Moreover, it has recently been found that polymorphism of WISP1 gene is related with the change of lung function in asthmatic subjects. Therefore, we hypothesized that WISP1 might be closely linked to occurrence and development of asthmatic airway remodeling. Aim of this study was to examine the roles of WISP1 in an asthmatic model with airway remodeling and assess the specific effects of WISP1 on human lung fibroblast in vitro. Animal models were developed by challenged with ovalbumin. The levels of WISP1 expression in animal models were assessed by real-time PCR and immunohistochemistry. To examine the specific effects of WISP1 on airway remodeling, WISP1 was depleted by neutralizing α-WISP1 antibodies in vivo. Moreover, human lung fibroblast (HFL-1) was challenged with WISP1 in the presence and absence of SH-5 in vitro. Our study showed that OVA exposure increased the levels of WISP1 expression in a rat asthma model. WISP1 depletion could partially inhibit OVA-induced airway remodeling. In vitro, WISP1-treated HFL-1 cells presented abnormal proliferation and over-expression of Col1a1 and Fn1. However, WISP1-induced collagen release from HFL-1 cells could be attenuated by pretreatment with an Akt inhibitor. Moreover, the levels of p-Akt and p-GSK-3β in WISP1-treated HFL-1 cells were also significantly elevated. In summary, WISP1 might initiate and perpetuate the pathological remodeling of asthma by inducing fibroblast proliferation and ECM deposition. The specific effects of WISP1 were likely due to activation of pulmonary Akt/GSK-3β signaling.

Sclerostin regulation of WISP1 and MMPS in chondrocytes is dependent on the LRP5/6 binding domain but not secretion from the cell

Sclerostin regulation of WISP1 and MMPS in chondrocytes is dependent on the LRP5/6 binding domain but not secretion from the cell

Genetic variations in the Wnt signaling pathway affect lung function in asthma patients

Identifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. The Wnt signal pathway plays a role in lung development and in asthma pathogenesis. However, whether genetic polymorphisms of Wnt signal pathway are associated with lung function in asthma patients remain unclear. We genotyped 2 single nucleotide polymorphisms (SNPs, rs2929973 and rs6581612) involved in the Wnt signal pathway in a cohort of 560 Chinese Han asthmatic children. Associations between each SNP and lung function, in a baseline exam, were tested using multiple linear regression models. We found that rs2929973 of the WISP1 gene was significantly associated with forced expiratory volume in 1 s (FEV1), with the G allele conferring significantly lower FEV1 values. However, the rs6581612 SNP of the WIF1 gene was not associated with differences in FEV1 values. We conclude that genetic variants in Wnt are associated with lung function and suggest that Wnt participates in inflammatory pathways that have an impact on the level of lung function.

Wnt1 Inducible Signaling Pathway Protein 1 (WISP1) Targets PRAS40 to Govern &amp;beta;-Amyloid Apoptotic Injury of Microglia

Given the present challenges to attain effective treatment for β-amyloid (Aβ) toxicity in neurodegenerative disorders such as Alzheimer's disease, development of novel cytoprotective pathways that can assist immune mediated therapies through the preservation of central nervous system microglia could offer significant promise. We show that the CCN4 protein, Wnt1 inducible signaling pathway protein 1 (WISP1), is initially up-regulated by Aβ and can modulate its endogenous expression for the protection of microglia during Aβ mediated apoptosis. WISP1 activates mTOR and phosphorylates p70S6K and 4EBP1 through the control of the regulatory mTOR component PRAS40. Loss of PRAS40 through gene reduction or inhibition by WISP1 is cytoprotective. WISP1 ultimately governs PRAS40 by sequestering PRAS40 intracellularly through post-translational phosphorylation and binding to protein 14-3-3. Our work identifies WISP1, mTOR signaling, and PRAS40 as targets for new strategies directed against Alzheimer's disease and related disorders.

EDITOR’S PERSPECTIVE [The Challenges for Drug Development: Cytokines, Genes, and Stem Cells

EDITOR’S PERSPECTIVE [The Challenges for Drug Development: Cytokines, Genes, and Stem Cells

Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition

The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype associated with chemotherapy resistance in epithelial cancers. However, the mechanisms underlying the EMT and its associated signaling dysfunctions are still poorly understood. In two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation, we investigated the status of several signaling elements downstream of G-protein receptors (GPR) and their functional roles in the invasive growth potential. We report that the E-cadherin repressors Slug, Zeb1/2 and Twist are overexpressed in these EMT cells characterized by a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the alternative GPR30 estrogen receptor and induction of the invasive growth in collagen type I gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. Accordingly, WISP-2 transcripts and Wisp-2 protein were depleted in these two convergent models of BC cell EMT. The EMT caused dominance of several proinvasive pathways downstream of GPR, including GαGβγ subunits, PKCα, AKT and c-Jun induction, constitutive activation of the actin-remodeling GTPase Rac1, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGβγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signaling independence to inhibitors of HER family tyrosine kinases and the mitogen- and stress-activated protein kinases. Our study suggests that the signaling protagonists GαGβγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.