Pancreatic cancer stem-like cells display aggressive behavior mediated via activation of FoxQ1.

Subpopulations of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) have been identified from most tumors, including pancreatic cancer (PC), and the existence of these cells is clinically relevant. Emerging evidence suggests that CSLCs participate in cell growth/proliferation, migration/invasion, metastasis, and chemo-radiotherapy resistance, ultimately contributing to poor clinical outcome. However, the pathogenesis and biological significance of CSLCs in PC has not been well characterized. In the present study, we found that isolated triple-marker-positive (CD44(+)/CD133(+)/EpCAM(+)) cells of human PC MiaPaCa-2 and L3.6pl cells behave as CSLCs. These CSLCs exhibit aggressive behavior, such as increased cell growth, migration, clonogenicity, and self-renewal capacity. The mRNA expression profiling analysis showed that CSLCs (CD44(+)/CD133(+)/EpCAM(+)) exhibit differential expression of more than 1,600 mRNAs, including FoxQ1, compared with the triple-marker-negative (CD44(-)/CD133(-)/EpCAM(-)) cells. The knockdown of FoxQ1 by its siRNA in CSLCs resulted in the inhibition of aggressive behavior, consistent with the inhibition of EpCAM and Snail expression. Mouse xenograft tumor studies showed that CSLCs have a 100-fold higher potential for tumor formation and rapid tumor growth, consistent with overexpression of CSC-associated markers/mediators, including FoxQ1, compared with its parental MiaPaCa-2 cells. The inhibition of FoxQ1 attenuated tumor formation and growth, and expression of CSC markers in the xenograft tumor derived from CSLCs of MiaPaCa-2 cells. These data clearly suggest the role of differentially expressed genes in the regulation of CSLC characteristics, further suggesting that targeting some of these genes could be important for the development of novel therapies for achieving better treatment outcome of PC.

Hyaluronic Acid Engineered Nanomicelles Loaded with 3,4-Difluorobenzylidene Curcumin for Targeted Killing of CD44+ Stem-Like Pancreatic Cancer Cells.

Cancer stem-like cells (CSLCs) play a pivotal role in acquiring multidrug resistant (MDR) phenotypes. It has been established that pancreatic cancers overexpressing CD44 receptors (a target of hyaluronic acid; HA) is one of the major contributors for causing MDR. Therefore, targeted killing of CD44 expressing tumor cells using HA based active targeting strategies may be beneficial for eradicating MDR-pancreatic cancers. Here, we report the synthesis of a new HA conjugate of copoly(styrene maleic acid) (HA-SMA) that could be engineered to form nanomicelles with a potent anticancer agent, 3,4-difluorobenzylidene curcumin (CDF). The anticancer activity of CDF loaded nanomicelles against MiaPaCa-2 and AsPC-1 human pancreatic cancer cells revealed dose-dependent cell killing. Results of cellular internalization further confirmed better uptake of HA engineered nanomicelles in triple-marker positive (CD44+/CD133+/EpCAM+) pancreatic CSLCs compared with triple-marker negative (CD44-/CD133-/EpCAM-) counterparts. More importantly, HA-SMA-CDF exhibited superior anticancer response toward CD44+ pancreatic CSLCs. Results further confirmed that triple-marker positive cells treated with HA-SMA-CDF caused significant reduction in CD44 expression and marked inhibition of NF-κB that in-turn can mitigate their proliferative and invasive behavior. Conclusively, these results suggest that the newly developed CD44 targeted nanomicelles may have great implications in treating pancreatic cancers including the more aggressive pancreatic CSLCs.

The biological kinship of hypoxia with CSC and EMT and their relationship with deregulated expression of miRNAs and tumor aggressiveness.

Hypoxia is one of the fundamental biological phenomena that are intricately associated with the development and aggressiveness of a variety of solid tumors. Hypoxia-inducible factors (HIF) function as a master transcription factor, which regulates hypoxia responsive genes and has been recognized to play critical roles in tumor invasion, metastasis, and chemo-radiation resistance, and contributes to increased cell proliferation, survival, angiogenesis and metastasis. Therefore, tumor hypoxia with deregulated expression of HIF and its biological consequence lead to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. It has been well recognized that cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotypic cells are associated with therapeutic resistance and contribute to aggressive tumor growth, invasion, metastasis and believed to be the cause of tumor recurrence. Interestingly, hypoxia and HIF signaling pathway are known to play an important role in the regulation and sustenance of CSCs and EMT phenotype. However, the molecular relationship between HIF signaling pathway with the biology of CSCs and EMT remains unclear although NF-κB, PI3K/Akt/mTOR, Notch, Wnt/ß-catenin, and Hedgehog signaling pathways have been recognized as important regulators of CSCs and EMT. In this article, we will discuss the state of our knowledge on the role of HIF-hypoxia signaling pathway and its kinship with CSCs and EMT within the tumor microenvironment. We will also discuss the potential role of hypoxia-induced microRNAs (miRNAs) in tumor development and aggressiveness, and finally discuss the potential effects of nutraceuticals on the biology of CSCs and EMT in the context of tumor hypoxia.

The immunological contribution of NF-κB within the tumor microenvironment: a potential protective role of zinc as an anti-tumor agent.

Over decades, cancer treatment has been mainly focused on targeting cancer cells and not much attention to host tumor microenvironment. Recent advances suggest that the tumor microenvironment requires in-depth investigation for understanding the interactions between tumor cell biology and immunobiology in order to optimize therapeutic approaches. Tumor microenvironment consists of cancer cells and tumor associated reactive fibroblasts, infiltrating non-cancer cells, secreted soluble factors or molecules, and non-cellular support materials. Tumor associated host immune cells such as Th(1), Th(2), Th17, regulatory cells, dendritic cells, macrophages, and myeloid-derived suppressor cells are major components of the tumor microenvironment. Accumulating evidence suggests that these tumor associated immune cells may play important roles in cancer development and progression. However, the exact functions of these cells in the tumor microenvironment are poorly understood. In the tumor microenvironment, NF-κB plays an important role in cancer development and progression because this is a major transcription factor which regulates immune functions within the tumor microenvironment. In this review, we will focus our discussion on the immunological contribution of NF-κB in tumor associated host immune cells within the tumor microenvironment. We will also discuss the potential protective role of zinc, a well-known immune response mediator, in the regulation of these immune cells and cancer cells in the tumor microenvironment especially because zinc could be useful for conditioning the tumor microenvironment toward innovative cancer therapy.

Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer-related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF-κB play a critical role in the development of PDAC in the compound K-Ras(G12D) and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K-Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real-time RT-PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K-Ras(G12D) expressing mice led to high expression of PDGF-D signaling pathway in the tumor and tumor-derived cell line (RInk-1 cells). Furthermore, PDGF-D knock-down in RInk-1 cells resulted in the inhibition of pancreatosphere formation and down-regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial-mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF-D, signaling.

Synthesis, characterization and anti-tumor activity of novel thymoquinone analogs against pancreatic cancer.

Thymoquinone (TQ), isolated from the seeds of Nigella sativa, show moderate efficacy against pancreatic cancer. In the present work we report synthesis and characterization of novel TQ analogs appended with gallate and fluorogallate pharmacophores and evaluation of their effects against pancreatic cancer cell lines for cell viability and induction of apoptosis. The efficacy of the analogs alone or in combination with Gemcitabine was assessed in vitro. LC-MS spectra of ATQTHB and ATQTFB showed major peaks corresponding to expected M+1 fragment at 316.34 and 322.34 respectively. Molecular docking studies revealed good fit for these analogs in the COX-2 protein cavity with better binding energies compared to parent TQ compound. Present TQ analogs exhibit superior anti-proliferative activity, excellent chemo-sensitizing activity against pancreatic cancer in vitro and in combination with Gemcitabine.

Anticancer action of garcinol in vitro and in vivo is in part mediated through inhibition of STAT-3 signaling.

Garcinol, obtained from Garcinia indica, has exhibited some promising anticancer activity. In particular, our earlier work has demonstrated its ability to inhibit cell proliferation and induction of apoptosis in multiple cancer cell lines representative of breast, prostate, as well as pancreatic cancers. However, its exact mechanism of action remains largely unclear. Here we show that garcinol also targets signal transducer and activator of transcription-3 (STAT-3) signaling pathway. STAT-3 is frequently found to be activated in many cancer types and this is the first report on such action of garcinol leading to its anticancer effects. Garcinol inhibited total, as well as phosphorylated, STAT-3 in breast, prostate and pancreatic cancer cell lines and was also found to inhibit cell invasion of all the cancer cell lines tested. STAT-3 phosphorylation was inhibited by garcinol in a dose-dependent manner. We also observed an inhibitory effect of garcinol on IL-6-induced STAT-3 phosphorylation and production of urokinase-type plasminogen activator, vascular endothelial growth factor and matrix metalloproteinase-9, which might explain the reduced invasion and aggressiveness of cells treated with garcinol. The results were further verified in vivo using MDA-MB-231 breast cancer mouse xenograft model where administration of garcinol significantly inhibited tumor growth, and western blot analysis of remnant tumor lysates showed reduced STAT-3 expression and activation. These results suggest that garcinol may have translational potential as chemopreventive or therapeutic agent against multiple cancers and inhibition of STAT-3 signaling pathway is one of the mechanisms by which garcinol exerts its anticancer effects.

The complexities of obesity and diabetes with the development and progression of pancreatic cancer.

Pancreatic cancer (PC) is one of the most lethal malignant diseases with the worst prognosis. It is ranked as the fourth leading cause of cancer-related deaths in the United States. Many risk factors have been associated with PC. Interestingly, large numbers of epidemiological studies suggest that obesity and diabetes, especially type-2 diabetes, are positively associated with increased risk of PC. Similarly, these chronic diseases (obesity, diabetes, and cancer) are also a major public health concern. In the U.S. population, 50 percent are overweight, 30 percent are medically obese, and 10 percent have diabetes mellitus (DM). Therefore, obesity and DM have been considered as potential risk factors for cancers; however, the focus of this article is restricted to PC. Although the mechanisms responsible for the development of these chronic diseases leading to the development of PC are not fully understood, the biological importance of the activation of insulin, insulin like growth factor-1 (IGF-1) and its receptor (IGF-1R) signaling pathways in insulin resistance mechanism and subsequent induction of compensatory hyperinsulinemia has been proposed. Therefore, targeting insulin/IGF-1 signaling with anti-diabetic drugs for lowering blood insulin levels and reversal of insulin resistance could be useful strategy for the prevention and/or treatment of PC. A large number of studies have demonstrated that the administration of anti-diabetic drugs such as metformin and thiazolidinediones (TZD) class of PPAR-γ agonists decreases the risk of cancers, suggesting that these agents might be useful anti-tumor agents for the treatment of PC. In this review article, we will discuss the potential roles of metformin and TZD anti-diabetic drugs as anti-tumor agents in the context of PC and will further discuss the complexities and the possible roles of microRNAs (miRNAs) in the pathogenesis of obesity, diabetes, and PC.

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer.

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.

Resveratrol-induced apoptosis is enhanced in low pH environments associated with cancer.

Many critical factors such as hypoxia, nutrient deficiency, activation of glycolytic pathway/Warburg effect contribute to the observed low pH in tumors compared to normal tissue. Studies suggest that such tumor specific acidic environment can be exploited for the development of therapeutic strategies against cancer. Independent observations show reduction in pH of mammalian cells undergoing internucleosomal DNA fragmentation and apoptosis. As such, our group has extensively demonstrated that anticancer mechanisms of different plant polyphenols involve mobilization of endogenous copper and consequent internucleosomal DNA breakage. Copper is redox active metal, an essential component of chromatin and is sensitive to subtle pH changes in its microenvironment. Here we explored whether, acidic pH promotes growth inhibition, apoptosis, and DNA damaging capacity of chemopreventive agent resveratrol. Our results reveal that growth inhibition and internucleosomal DNA fragmentation induced apoptosis in Capan-2 and Panc-28 pancreatic cancer cell lines (and not in normal HPDE cells) by resveratrol is enhanced at lower pH. Using comet assay, we further demonstrate that DNA breakage by resveratrol is enhanced with acidification. Membrane permeable copper specific chelator neocuproine (and not iron chelator orthophenanthroline) abrogated growth inhibition and apoptosis by resveratrol. Western blot results show enhanced activation of DNA laddering marker H2.aX by resveratrol at acidic pH that was reversed by neocuproine and not by orthophenanthroline. Our findings provide irrevocable proof that low pH environment can be turned into tumor weakness and assist in eradication of cancer cells by resveratrol.

Inclusion complex of novel curcumin analogue CDF and ß-cyclodextrin (1:2) and its enhanced in vivo anticancer activity against pancreatic cancer.

PURPOSE: Several formulations have been proposed to improve the systemic delivery of novel cancer therapeutic compounds, including cyclodextrin derivatives. We aimed to synthesize and characterize of CDF-ß-cyclodextrin inclusion complex (1:2) (CDFCD). METHODS: The compound was characterized by Fourier transform infrared, differential scanning calorimetry, powder X-ray diffraction studies, H1 & C13 NMR studies and scanning electron microscopic analysis. Its activity was tested against multiple cancer cell lines, and in vivo bioavailability was checked. RESULTS: CDF-ß-cyclodextrin was found to lower IC(50) value by half when tested against multiple cancer cell lines. It preferentially accumulated in the pancreas, where levels of CDF-ß-cyclodextrin in mice were 10 times higher than in serum, following intravenous administration of an aqueous CDF-ß-cyclodextrin preparation. CONCLUSIONS: Novel curcumin analog CDF preferentially accumulates in the pancreas, leading to its potent anticancer activity against pancreatic cancer cells. Synthesis of such CDF-ß-cyclodextrin self-assembly is an effective strategy to enhance its bioavailability and tissue distribution, warranting further evaluation for CDF delivery in clinical settings for treatment of human malignancies.

Targeting Notch signaling pathway to overcome drug resistance for cancer therapy.

Chemotherapy is an important therapeutic strategy for cancer treatment and remains the mainstay for the management of human malignancies; however, chemotherapy fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Recently, emerging evidences suggest that Notch signaling pathway is one of the most important signaling pathways in drug-resistant tumor cells. Moreover, down-regulation of Notch pathway could induce drug sensitivity, leading to increased inhibition of cancer cell growth, invasion, and metastasis. This article will provide a brief overview of the published evidences in support of the roles of Notch in drug resistance and will further summarize how targeting Notch by "natural agents" could become a novel and safer approach for the improvement of tumor treatment by overcoming drug resistance.

Emerging roles of PDGF-D signaling pathway in tumor development and progression.

Platelet-derived growth factor-D (PDGF-D) can regulate many cellular processes, including cell proliferation, apoptosis, transformation, migration, invasion, angiogenesis and metastasis. Therefore PDGF-D signaling has been considered to be important in human malignancies, and thus PDGF-D signaling may represent a novel therapeutic target, and as such suggests that the development of agents that will target PDGF-D signaling is likely to have a significant therapeutic impact on human cancers. This mini-review describes the mechanisms of signal transduction associated with PDGF-D signaling to support the role of PDGF-D in the carcinogenesis. Moreover, we summarize data on several PDGF-D inhibitors especially naturally occurring "chemopreventive agent" such an indole compound, which we believe could serve as a novel agent for the prevention of tumor progression and/or treatment of human malignancies by targeted inactivation of PDGF-D signaling.

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells.

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b, and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.

Down-regulation of Notch-1 is associated with Akt and FoxM1 in inducing cell growth inhibition and apoptosis in prostate cancer cells.

Although many studies have been done to uncover the mechanisms by which down-regulation of Notch-1 exerts its anti-tumor activity against a variety of human malignancies, the precise molecular mechanisms remain unclear. In the present study, we investigated the cellular consequence of Notch-1 down-regulation and also assessed the molecular consequence of Notch-1-mediated alterations of its downstream targets on cell viability and apoptosis in prostate cancer (PCa) cells. We found that the down-regulation of Notch-1 led to the inhibition of cell growth and induction of apoptosis, which was mechanistically linked with down-regulation of Akt and FoxM1, suggesting for the first time that Akt and FoxM1 are downstream targets of Notch-1 signaling. Moreover, we found that a "natural agent" (genistein) originally discovered from soybean could cause significant reduction in cell viability and induced apoptosis of PCa cells, which was consistent with down-regulation of Notch-1, Akt, and FoxM1. These results suggest that down-regulation of Notch-1 by novel agents could become a newer approach for the prevention of tumor progression and/or treatment, which is likely to be mediated via inactivation of Akt and FoxM1 signaling pathways in PCa.

Restoring sensitivity to oxaliplatin by a novel approach in gemcitabine-resistant pancreatic cancer cells in vitro and in vivo.

Oxaliplatin (OxP) has been used in combination therapy with gemcitabine for the treatment of pancreatic cancer (PC), but the beneficial effect was marginal, which is believed to be due to de novo and acquired drug resistance of PC. Here, we report our in vitro and in vivo preclinical evidence in support of chemosensitization of drug-resistant cells by a nontoxic chemopreventive agent (genistein). Genistein pretreatment together with low concentration of OxP showed significant reduction in cell viability and colony formation concomitant with increased apoptosis (p < 0.01), which was highly synergistic. Drug resistance of PC is allegedly linked with both constitutive and OxP-induced activation of NF-κB, and we found that inactivation of (nuclear factor kappa B) NF-κB by genistein before treatment of cells with OxP was required for cell killing, which was consistent with the downregulation of NF-κB and its downstream antiapoptotic genes (Bcl-2, XIAPs and survivin). Most importantly, our in vivo experiments using orthotopic mouse model showed significant reduction in tumor size (p < 0.01) and reduction of locoregional lymph node metastasis by combination treatment. These results were also consistent with inactivation of NF-κB and the downregulation of NF-κB downstream genes, decreased proliferation marker (Ki-67) and increased apoptosis (TUNEL) in tumor remnants, all of which was consistent with in vitro findings. From these results, we conclude that genistein sensitizes drug-resistant PC to OxP, which is mechanistically linked with inactivation of NF-κB signaling, resulting in greater antitumor effects, and thus our data suggest that this approach could be useful in improving the treatment outcome for patients diagnosed with PC.

Curcumin enhances dasatinib-induced inhibition of growth and transformation of colon cancer cells.

Colorectal cancer is the third most common form of malignancy, behind prostate and lung cancers. Despite recent advances in medicine, mortality from colorectal cancer remains high, highlighting the need for improved therapies. Numerous studies have demonstrated increased activation of EGFR and its family members (EGFRs), IGF-1R as well as c-Src in colorectal cancer. The current study was undertaken to examine the effectiveness of combination therapy of dasatinib (BMS-354825; Bristol-Myers Squibb), a highly specific inhibitor of Src family kinases (SFK) and a nontoxic dietary agent; curcumin (diferuloylmethane), in colorectal cancer in in vitro and in vivo experimental models. For the latter, we utilized C57BL/6 APC(Min+/-) mice. Initial in vitro studies revealed synergistic interactions between the two agents. Additionally, we have observed that combination treatment causes a much greater inhibition of the following metastatic processes than either agent alone: (i) colony formation, (ii) invasion through extracellular matrix and (iii) tubule formation by endothelial cells. Dasatinib affects the cell adhesion phenotype of colon cancer HCT-116 cells whereas the combination therapy enhances this effect to a greater extent. Preclinical investigation revealed that the combination therapy to be highly effective causing an over 95% regression of intestinal adenomas in Apc(Min+/-) mice, which could be attributed to decreased proliferation and increased apoptosis. In conclusion, our data suggest that combination treatment of dasatinib and curcumin could be a potential therapeutic strategy for colorectal cancer.

Platelet-derived growth factor-D contributes to aggressiveness of breast cancer cells by up-regulating Notch and NF-κB signaling pathways.

Platelet-derived growth factor-D (PDGF-D) has been linked with several human malignancies; however, its role in breast cancer progression is not known. We found that PDGF-D expressing breast cancer cell lines MDA-MB-231 and SUM-149 are more invasive compared to cell lines with little or no expression of PDGF-D such as MDA-MB-468 and MCF-7 cells. Over-expression of PDGF-D in PDGF-D low expressing MDA-MB-468 and MCF-7 cells by cDNA transfection showed increased cell proliferation while silencing the expression of PDGF-D by siRNA in PDGF-D high expressing MDA-MB-231 and SUM-149 cells showed decreased cell proliferation and increased apoptosis. Moreover, PDGF-D over-expression was positively correlated with the expression of Notch-1 and Jagged-1, and the expression of mesenchymal markers (Vimentin and ZEB-2) with concomitant decreased expression of epithelial marker E-cadherin. Since NF-κB activation plays a crucial role in Notch signaling as well as in epithelial-mesenchymal transition and tumor aggressiveness, we determined the DNA binding activity of NF-κB and our findings are consistent showing that PDGF-D over-expression led to increased DNA binding activity of NF-κB while it was found to be decreased by inactivation of PDGF-D. These results were also consistent with the expression and activity of MMP-9 and VEGF, as well as invasive characteristics. Further, forced expression of Notch-1/Jagged-1 by cDNA transfection de-repressed the effects of PDGF-D silencing on NF-κB activity and invasion. From these results, we conclude that PDGF-D plays an important role in breast tumor aggressiveness and this process is mechanistically linked with the activation of Notch and NF-κB signaling.