Yadwinder S. Deol

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.

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.

HIV-1 gp120-induced migration of dendritic cells is regulated by a novel kinase cascade involving Pyk2, p38 MAP kinase, and LSP1

Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2-inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogen-activated protein kinase, which in turn activates the F-actin-binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person.

Tumor-suppressive effects of psoriasin (S100A7) are mediated through the β-catenin/T cell factor 4 protein pathway in estrogen receptor-positive breast cancer cells.

Background: The role of S100A7 in ERα+ breast cancer is not known. Results: S100A7 overexpression in ERα+ breast cancer cells inhibits tumorigenesis by decreasing β-catenin and TCF4 expression through activation of GSK3β and E-cadherin. Conclusion: S100A7 may regulate tumorigenesis through modulation of the β-catenin/TCF4 pathway. Significance: S100A7 may possess differential activity in ERα+ compared with ERα− cells, as S100A7 has been shown to enhance tumorigenesis in ERα− cells. Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα−) invasive breast cancers, its role in ERα-positive (ERα+) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα+ breast cancer cells and observed that S100A7 overexpression in ERα+ breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα+ breast cancer cells. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα+ breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα+ breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the β-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα− cells, our studies suggest that S100A7 may possess differential activities in ERα+ compared with ERα− cells.

Differential role of psoriasin (S100A7) in estrogen receptor α positive and negative breast cancer cells occur through actin remodeling.

Psoriasin (S100A7) is a calcium-binding protein that has shown to be highly expressed in high-grade ductal carcinoma in situ (DCIS) and a subset of invasive breast cancers. However, its role in invasion and metastasis is not very well known. In this study, we have shown that S100A7 differentially regulates epidermal growth factor (EGF)-induced cell migration and invasion in ERα− MDA-MB-231 cells and ERα+ MCF-7 and T47D breast cancer cells. Further signaling studies revealed that S100A7 enhances EGF-induced EGFR phosphorylation and actin remodeling that seems to favor lamellipodia formation in ERα− cells. In addition, S100A7 overexpression enhanced NF-κB-mediated matrix metalloproteinase-9 (MMP-9) secretion in MDA-MB-231 cells indicating its role in enhanced invasiveness. However, S100A7 overexpression inhibited migration and invasion of MCF-7 cells by inactivating Rac-1 pathway and MMP-9 secretion. Moreover, S100A7 overexpressing MDA-MB-231 cells showed enhanced metastasis compared to vector control in in vivo nude mice as detected by bioluminescence imaging. Our tissue microarray data also revealed predominant expression of S100A7 in ERα− metastatic carcinoma, especially in lymph node regions. Overall these studies suggest that S100A7 may enhance metastasis in ERα− breast cancer cells by a novel mechanism through regulation of actin cytoskeleton and MMP-9 secretion.

miR-29b defines the pro-/anti-proliferative effects of S100A7 in breast cancer

IntroductionS100A7 (Psoriasin) is an inflammatory protein known to be upregulated in breast cancer. However, the role of S100A7 in breast cancer has been elusive, since both pro- and anti-proliferative roles have been reported in different types of breast cancer cells and animal models. To date, the mechanism by which S100A7 differentially regulates breast cancer cell proliferation is still not clear.MethodsWe used Gene Functional Enrichment Analysis to search for the determining factor of S100A7 differential regulation. We confirmed the factor and elaborated its regulating mechanism using in vitro cell culture. We further verified the findings using xenografts of human breast cancer cells in nude mice.ResultsIn the present study, we show that S100A7 significantly upregulates the expression of miR-29b in Estrogen Receptor (ER)-positive breast cancer cells (represented by MCF7), and significantly downregulates miR-29b in ER-negative cells (represented by MDA-MB-231). The differential regulation of miR-29b by S100A7 in ER-positive and ER-negative breast cancer is supported by the gene expression analysis of TCGA invasive breast cancer dataset. miR-29b transcription is inhibited by NF-κB, and NF-κB activation is differentially regulated by S100A7 in ER-positive and ER-negative breast cancer cells. This further leads to differential regulation of PI3K p85α and CDC42 expression, p53 activation and p53-associated anti-proliferative pathways. Reversing the S100A7-caused changes of miR-29b expression by transfecting exogenous miR-29b or miR-29b-Decoy can inhibit the effects of S100A7 on in vitro cell proliferation and tumor growth in nude mice.ConclusionsThe distinct modulations of the NF-κB – miR-29b – p53 pathway make S100A7 an oncogene in ER-negative and a cancer-suppressing gene in ER-positive breast cancer cells, with miR-29b being the determining regulatory factor.

Role of CXCL12 and CXCR4 in Tumor Biology and Metastasis

Chemokine receptor CXCR4 and its cognate ligand CXCL12, also known as stromal derived factor-1 (SDF-1), have been shown to play an important role in growth and metastasis of various tumors. CXCR4 is the most common chemokine receptor that has been demonstrated to be overexpressed in several cancers. Its overexpression is also correlated with poor clinical outcomes and survival in various cancers, including breast, prostate, and lung. CXCR4/CXCL12 signaling axis plays an important role in organ selective metastasis since CXCR4 overexpressing cancer cells have been shown to metastasize to organs such as bone, lymph nodes, liver, lung, and brain, which produce high amounts of CXCL12 and thus provide a favorable microenvironment. CXCR4/CXCL12 axis has been shown to enhance tumor growth and metastasis by maintaining cancer stem cells and modulating tumor stroma through activation of cancer-associated fibroblasts and recruitment of CXCR4+ endothelial precursor cells, thereby enhancing angiogenesis. CXCR4/CXCL12 axis has been shown to mediate both pro-tumorigenesis and metastasis by activating multiple signaling pathways, including protein tyrosine pathways through activation of Pyk2. Several CXCR4/CXCL12 antagonists/agonists have been shown to have potential therapeutic effects as they significantly inhibit tumor growth and metastasis of various cancers in both in vitro and in vivo mouse models. These studies suggest that the CXCR4/CXCL12 signaling axis is an important target for developing innovative therapies against various cancers.

Abstract 1593: Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients

Introduction and Objective: A necessary step in distant metastasis is the hematogenous dissemination of cancer cells from the primary tumor site to remote sites. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival in many solid malignancies. Immune-affinity based capture is the most commonly used method for the isolation of CTCs which utilizes antibodies to capture tumor cells expressing specific proteins. However, immune-affinity based approaches offer low throughput (∼1mL/hr) and considerable cell loss (∼20-40%) resulting from heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing physical properties of CTCs have been developed to overcome the limitations of immune-affinity based isolation techniques, including micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs that yields the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. Methods: The PDMS-made microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. The device was optimized with MCF-7 and Panc-1 cell lines spiked into PBS buffer and also diluted blood. It was then tested on 10 mL blood samples from patients with metastatic breast cancer. The separated cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, CD45 negative cells. Results: Samples were processed through the inertial microfluidic device that enriches CTCs in the second outlet based on size difference between CTCs and blood cells. The device was optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (92% for both spiked samples of MCF-7 and PANC-1 cell lines) and high white blood cells (WBCs) removal (91%). To determine the efficiency of capture of rare cell populations, healthy donor blood samples were spiked with MCF-7/GFP at 100 cells and 95% recovery was obtained. In patient samples, we identified CTCs in 24 of 27 (89%) breast patients with metastatic disease (4.3±4.8 CTCs/mL) with low WBCs contamination (465±473 WBCs/mL). Conclusion: The study of CTCs could have a direct impact upon patient care by presenting a novel CTC isolation method. This technology may be applicable to early detection, and also for monitoring response to treatment. Our approach is superior to current strategies because it is independent of cell surface markers, which may be varied in tumor cells, and may exclude cancer stem cells. Citation Format: Eric Lin, Lianette Rivera, Hyeun Joong Yoon, Shamileh Fouladdel, Jacob Wieger, Stephanie Guthrie, Yadwinder S. Deol, Shawn G. Clouthier, Tahra K. Luther, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1593. doi:10.1158/1538-7445.AM2015-1593

Abstract 388: mS100a7a15 enhances mammary tumor growth and metastasis by recruiting tumor associated macrophages

mS100a7a15 is the murine ortholog of human S100A7 and S100A15 proteins. Both S100A7 and S100A15 have been shown to play an important role in breast cancer. S100A7 has been shown to be highly expressed in high grade ductal carcinoma in situ and invasive breast cancers. Its expression is also related to poor prognosis and associated with increased inflammatory infiltrates and various inflammatory disorders. However, the exact mechanism by which S100A7 or S100A15 enhances breast cancer growth is not known. In the present study, we determined the molecular mechanisms by which mS100a7a15 enhances growth by overexpressing mS100a7a15 in MDA-MB-231 cells. We showed that mS100a7a15 enhances expression of proinflammatory molecules CXCL1 and CXCL8. In addition, we observed that supernatants obtained from mS100a7a15 overexpressing cells enhanced chemotaxis of murine RAW264.7 macrophages. Further elucidation revealed that mS100a7/a15 mediates its effects by binding to receptor for advanced glycation end products (RAGE). We further analyzed the role of mS100a7a15 on modulation of tumor growth and inflammatory pathways in breast cancer by generating inducible bi-transgenic MMTV-rtTA; mS100a7a15 mice (MMTV-mS100a7a15). These mice showed enhanced mS100a7a15 protein expression upon doxycycline treatment. Mammary glands isolated from these mice showed enhanced hyperplasia upon doxycycline treatment for 3 months compared to uninduced mice. Further studies revealed enhanced recruitment of macrophages in mammary glands and activation of STAT3 in induced mice. Orthotopic implantation of MVT-1 breast tumor cells (derived from MMTV-c-Myc; MMTV-VEGF mice) into the mammary glands of these mice showed enhanced tumor growth and metastasis in doxycycline treated mice compared to the control. Tumors and lung tissues obtained from these mice showed enhanced pro-metastatic gene expression and recruitment of F4/80 and CD206 positive M2 tumor-associated macrophages (TAM). However, no difference was observed in CD3 + and CD4 + T lymphocytes. Further elucidation of the role of macrophages by in vivo depletion of macrophages using clodronate liposomes revealed that mS100a7a15-mediated recruitment of TAM is important for tumor growth, angiogenesis and metastasis. Furthermore, mice treated with STAT3 inhibitors showed reduced mS100a7a15-induced hyperplasia. STAT3 has been shown to regulate expression of various inflammatory molecules. These studies using a novel mS100a7a15 bi-transgenic model system demonstrate that mS100a7a15 enhances breast tumor growth and metastasis by enhancing inflammatory signals that result in enhanced recruitment of TAM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 388. doi:1538-7445.AM2012-388

Abstract 403: Overexpression of S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective: Psoriasin (S100A7), small 11 kDa calcium binding protein, has been shown to be highly expressed in ER-alpha-negative (ERα−) invasive breast carcinomas with poor patient outcome. However, the role of S100A7 in breast cancer progression and metastasis is not well understood. Here we elucidate S100A7-mediated effects in breast tumorogenesis. Methods: We studied EGF-mediated cell migration and cell motility in vitro in S100A7 overexpressing MDA-MB-231 (231-S100A7) cells by standard assays. We generated bi-transgenic MMTV-rtTA; mS100A7/A15 mice which expressed mS100A7/A15 in mammary glands under doxycycline (Dox) (1g/kg) treatment. Mammary glands were isolated and stained for carmine, HE MMTV-VEGF) orthotopically into the mammary glands of bi-transgenic mice to study the role of mS100A7/A15 in mammary tumor growth and metastasis. Results: In the present investigation, we have shown that S100A7 overexpression in ERα− breast cancer cell line enhanced EGF-induced migration and wound healing compared to vector control cells. Further elucidation of signaling mechanisms reveal that S100A7 enhanced EGF-induced EGFR and Stat3 activation. Microarray and real time PCR analysis revealed upregulation of pro-inflammatory molecules such as CXCL8, CXCL1, IL-1α, serum amyloid A2 (SAA2) and MMP9 in 231-S100A7 cells compared to 231-Vec. These results were further confirmed with cytokine array analysis in the conditioned media (CM) of 231-S100A7 cells compared to 231-Vec. To further characterize the role of S100A7, we targeted the expression of murine homologue of S100A7, also known as mS100A7/A15, to mammary epithelial cells by generating bi-transgenic MMTV-rtTA; mS100A7/A15 mice. Morphological examination of the mammary glands after three months of Dox treatment revealed ductal hyperplasia, enhanced mS100A7/A15, Ki67 and cyclin D1 expression compared to uninduced mice. We also showed enhanced activation of Stat3, AKT, ERK1/2, MMP2 and SAA2 in the mammary glands of these mice. We further observed enhanced tumor growth and metastasis of orthotopically injected MVT1 cells in Dox treated compared to untreated mice. Tumors and lung tissues obtained from Dox treated bi-transgenic mice showed increased expression of prometastatic genes and enhanced recruitment of tumor-associated macrophages (TAM). Furthermore, CM from 231-S100A7 cells significantly enhanced migration of THP1-differentiated macrophages. Conclusion: These findings suggest that S100A7 may regulate breast cancer progression and metastasis by enhancing inflammatory signals that result in enhanced recruitment of TAM. Furthermore, these studies suggest that S100A7 is a novel therapeutic target for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 403. doi:10.1158/1538-7445.AM2011-403