Mohd W. Nasser

MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to Sorafenib

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.

Down-regulation of Micro-RNA-1 (miR-1) in Lung Cancer SUPPRESSION OF TUMORIGENIC PROPERTY OF LUNG CANCER CELLS AND THEIR SENSITIZATION TO DOXORUBICIN-INDUCED APOPTOSIS BY miR-1

Micro-RNAs are ∼21–25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPα, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.

Synthetic cannabinoid receptor agonists inhibit tumor growth and metastasis of breast cancer

Cannabinoids have been reported to possess antitumorogenic activity. Not much is known, however, about the effects and mechanism of action of synthetic nonpsychotic cannabinoids on breast cancer growth and metastasis. We have shown that the cannabinoid receptors CB1 and CB2 are overexpressed in primary human breast tumors compared with normal breast tissue. We have also observed that the breast cancer cell lines MDA-MB231, MDA-MB231-luc, and MDA-MB468 express CB1 and CB2 receptors. Furthermore, we have shown that the CB2 synthetic agonist JWH-133 and the CB1 and CB2 agonist WIN-55,212-2 inhibit cell proliferation and migration under in vitro conditions. These results were confirmed in vivo in various mouse model systems. Mice treated with JWH-133 or WIN-55,212-2 showed a 40% to 50% reduction in tumor growth and a 65% to 80% reduction in lung metastasis. These effects were reversed by CB1 and CB2 antagonists AM 251 and SR144528, respectively, suggesting involvement of CB1 and CB2 receptors. In addition, the CB2 agonist JWH-133 was shown to delay and reduce mammary gland tumors in the polyoma middle T oncoprotein (PyMT) transgenic mouse model system. Upon further elucidation, we observed that JWH-133 and WIN-55,212-2 mediate the breast tumor-suppressive effects via a coordinated regulation of cyclooxygenase-2/prostaglandin E2 signaling pathways and induction of apoptosis. These results indicate that CB1 and CB2 receptors could be used to develop novel therapeutic strategies against breast cancer growth and metastasis. [Mol Cancer Ther 2009;8(11):3117–29]

S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways

S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways.

Cannabinoid Receptors, CB1 and CB2, as Novel Targets for Inhibition of Non–Small Cell Lung Cancer Growth and Metastasis

Non–small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide; however, only limited therapeutic treatments are available. Hence, we investigated the role of cannabinoid receptors, CB1 and CB2, as novel therapeutic targets against NSCLC. We observed expression of CB1 (24%) and CB2 (55%) in NSCLC patients. Furthermore, we have shown that the treatment of NSCLC cell lines (A549 and SW-1573) with CB1/CB2- and CB2-specific agonists Win55,212-2 and JWH-015, respectively, significantly attenuated random as well as growth factor-directed in vitro chemotaxis and chemoinvasion in these cells. We also observed significant reduction in focal adhesion complex, which plays an important role in migration, upon treatment with both JWH-015 and Win55,212-2. In addition, pretreatment with CB1/CB2 selective antagonists, AM251 and AM630, prior to JWH-015 and Win55,212-2 treatments, attenuated the agonist-mediated inhibition of in vitro chemotaxis and chemoinvasion. In addition, both CB1 and CB2 agonists Win55,212-2 and JWH-133, respectively, significantly inhibited in vivo tumor growth and lung metastasis (∼50%). These effects were receptor mediated, as pretreatment with CB1/CB2 antagonists abrogated CB1/CB2 agonist–mediated effects on tumor growth and metastasis. Reduced proliferation and vascularization, along with increased apoptosis, were observed in tumors obtained from animals treated with JWH-133 and Win55,212-2. Upon further elucidation into the molecular mechanism, we observed that both CB1 and CB2 agonists inhibited phosphorylation of AKT, a key signaling molecule controlling cell survival, migration, and apoptosis, and reduced matrix metalloproteinase 9 expression and activity. These results suggest that CB1 and CB2 could be used as novel therapeutic targets against NSCLC. Cancer Prev Res; 4(1); 65–75. ©2010 AACR.

RAGE Mediates S100A7-Induced Breast Cancer Growth and Metastasis by Modulating the Tumor Microenvironment

RAGE is a multifunctional receptor implicated in diverse processes including inflammation and cancer. In this study, we report that RAGE expression is upregulated widely in aggressive triple-negative breast cancer (TNBC) cells, both in primary tumors and in lymph node metastases. In evaluating the functional contributions of RAGE in breast cancer, we found that RAGE-deficient mice displayed a reduced propensity for breast tumor growth. In an established model of lung metastasis, systemic blockade by injection of a RAGE neutralizing antibody inhibited metastasis development. Mechanistic investigations revealed that RAGE bound to the proinflammatory ligand S100A7 and mediated its ability to activate ERK, NF-κB, and cell migration. In an S100A7 transgenic mouse model of breast cancer (mS100a7a15 mice), administration of either RAGE neutralizing antibody or soluble RAGE was sufficient to inhibit tumor progression and metastasis. In this model, we found that RAGE/S100A7 conditioned the tumor microenvironment by driving the recruitment of MMP9-positive tumor-associated macrophages. Overall, our results highlight RAGE as a candidate biomarker for TNBCs, and they reveal a functional role for RAGE/S100A7 signaling in linking inflammation to aggressive breast cancer development.

Crosstalk between Chemokine Receptor CXCR4 and Cannabinoid Receptor CB2 in Modulating Breast Cancer Growth and Invasion

Background Cannabinoids bind to cannabinoid receptors CB1 and CB2 and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB2 may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis. Methodology/Principal Findings We observed high expression of both CB2 and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB2-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems. Conclusions/Significance This study provides novel insights into the crosstalk between CB2 and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB2 receptors could be used for developing innovative therapeutic strategies against breast cancer.

CXCR3 deficiency enhances tumor progression by promoting macrophage M2 polarization in a murine breast cancer model.

Tumor associated macrophages play a vital role in determining the outcome of breast cancer. We investigated the contribution of the chemokine receptor CXCR3 to antitumor immune responses using a cxcr3 deficient mouse orthotopically injected with a PyMT breast cancer cell line. We observed that cxcr3 deficient mice displayed increased IL‐4 production and M2 polarization in the tumors and spleens compared to WT mice injected with PyMT cells. This was accompanied by larger tumor development in cxcr3−/− than in WT mice. Further, tumor‐promoting myeloid derived immune cell populations accumulated in higher proportions in the spleens of cxcr3 deficient mice. Interestingly, cxcr3−/− macrophages displayed a deficiency in up‐regulating inducible nitric oxide synthase after stimulation by either IFN‐γ or PyMT supernatants. Stimulation of bone marrow derived macrophages by PyMT supernatants also resulted in greater induction of arginase‐1 in cxcr3−/− than WT mice. Further, cxcr3−/− T cells activated with CD3/CD28 in vitro produced greater amounts of IL‐4 and IL‐10 than T cells from WT mice. Our data suggests that a greater predisposition of cxcr3 deficient macrophages towards M2 polarization contributes to an enhanced tumor promoting environment in cxcr3 deficient mice. Although CXCR3 is known to be expressed on some macrophages, this is the first report that demonstrates a role for CXCR3 in macrophage polarization and subsequent breast tumor outcomes. Targeting CXCR3 could be a potential therapeutic approach in the management of breast cancer tumors.

C-X-C motif chemokine 12/C-X-C chemokine receptor type 7 signaling regulates breast cancer growth and metastasis by modulating the tumor microenvironment.

IntroductionAlthough C-X-C motif chemokine 12 (CXCL12) has been shown to bind to C-X-C chemokine receptor type 7 (CXCR7), the exact molecular mechanism regulations by CXCL12/CXCR7 axis in breast tumor growth and metastasis are not well understood. CXCR7 expression has been shown to be upregulated during pathological processes such as inflammation and cancer.MethodsBreast cancer cell lines were genetically silenced or pharmacologically inhibited for CXCR7 and/or its downstream target signal transducer and activator of transcription 3 (STAT3). 4T1 or 4T1 downregulated for CXCR7 and 4T1.2 breast cancer cell lines were injected in mammary gland of BALB/c mice to form tumors, and the molecular pathways regulating tumor growth and metastasis were assessed.ResultsIn this study, we observed that CXCL12 enhances CXCR7-mediated breast cancer migration. Furthermore, genetic silencing or pharmacologic inhibition of CXCR7 reduced breast tumor growth and metastasis. Further elucidation of mechanisms revealed that CXCR7 mediates tumor growth and metastasis by activating proinflammatory STAT3 signaling and angiogenic markers. Furthermore, enhanced breast tumorigenicity and invasiveness were associated with macrophage infiltration. CXCR7 recruits tumor-promoting macrophages (M2) to the tumor site through regulation of the macrophage colony-stimulating factor (M-CSF)/macrophage colony-stimulating factor receptor (MCSF-R) signaling pathway. In addition, CXCR7 regulated breast cancer metastasis by enhancing expression of metalloproteinases (MMP-9, MMP-2) and vascular cell-adhesion molecule-1 (VCAM-1). We also observed that CXCR7 is highly expressed in invasive ductal carcinoma (IDC) and metastatic breast tissue in human patient samples. In addition, high CXCR7 expression in tumors correlates with worse prognosis for both overall survival and lung metastasis-free survival in IDC patients.ConclusionThese observations reveal that CXCR7 enhances breast cancer growth and metastasis via a novel pathway by modulating the tumor microenvironment. These findings identify CXCR7-mediated STAT3 activation and modulation of the tumor microenvironment as novel regulation of breast cancer growth and metastasis. These studies indicate that new strategies using CXCR7 inhibitors could be developed for antimetastatic therapy.

Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway: Novel anti‐tumor mechanisms of Cannabidiol in breast cancer

The anti‐tumor role and mechanisms of Cannabidiol (CBD), a non‐psychotropic cannabinoid compound, are not well studied especially in triple‐negative breast cancer (TNBC). In the present study, we analyzed CBDs anti‐tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here ‐for the first time‐that CBD significantly inhibits epidermal growth factor (EGF)‐induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF‐induced activation of EGFR, ERK, AKT and NF‐kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor‐associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD‐treated cancer cells. The conditioned medium of CBD‐treated cancer cells also showed lower levels of GM‐CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows ‐for the first time‐that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.