Hidayatullah G. Munshi

TGF-β: Duality of Function Between Tumor Prevention and Carcinogenesis

Several mechanisms underlying tumor progression have remained elusive, particularly in relation to transforming growth factor beta (TGF-β). Although TGF-β initially inhibits epithelial growth, it appears to promote the progression of advanced tumors. Defects in normal TGF-β pathways partially explain this paradox, which can lead to a cascade of downstream events that drive multiple oncogenic pathways, manifesting as several key features of tumorigenesis (uncontrolled proliferation, loss of apoptosis, epithelial-to-mesenchymal transition, sustained angiogenesis, evasion of immune surveillance, and metastasis). Understanding the mechanisms of TGF-β dysregulation will likely reveal novel points of convergence between TGF-β and other pathways that can be specifically targeted for therapy.

Crosstalk between Mast Cells and Pancreatic Cancer Cells Contributes to Pancreatic Tumor Progression

Purpose: To assess the clinical and pathologic significance of mast cell infiltration in human pancreatic cancer and evaluate crosstalk between mast cells and cancer cells in vitro. Experimental Design: Immunohistochemistry for tryptase was done on 53 pancreatic cancer specimens. Mast cell counts were correlated with clinical variables and survival. Serum tryptase activity from patients with cancer was compared with patients with benign pancreatic disease. In vitro, the effect of pancreatic cancer–conditioned medium on mast cell migration was assessed. The effect of conditioned medium from the human mast cell line, LAD-2, on cancer and normal ductal cell proliferation was assessed by thymidine incorporation. Matrigel invasion assays were used to evaluate the effect of mast cell–conditioned medium on cancer cell invasion in the presence and absence of a matrix metalloproteinase inhibitor, GM6001. Results: Mast cell infiltration was significantly increased in pancreatic cancer compared with normal pancreatic tissue (11.4 ± 6.7 versus 2.0 ± 1.4, P < 0.001). Increased infiltrating mast cells correlated with higher grade tumors (P < 0.0001) and worse survival. Patients with pancreatic cancer had elevated serum tryptase activity (P < 0.05). In vitro, AsPC1 and PANC-1 cells induced mast cell migration. Mast cell–conditioned medium induced pancreatic cancer cell migration, proliferation, and invasion but had no effect on normal ductal cells. Furthermore, the effect of mast cells on cancer cell invasion was, in large part, matrix metalloproteinase–dependent. Conclusions: Tumor-infiltrating mast cells are associated with worse prognosis in pancreatic cancer. In vitro, the interaction between mast cells and pancreatic cancer cells promotes tumor growth and invasion. Clin Cancer Res; 16(8); 2257–65. ©2010 AACR.

Biochemical role of the collagen-rich tumour microenvironment in pancreatic cancer progression

PDAC (pancreatic ductal adenocarcinoma) is among the most deadly of human malignances. A hallmark of the disease is a pronounced collagen-rich fibrotic extracellular matrix known as the desmoplastic reaction. Intriguingly, it is precisely these areas of fibrosis in which human PDAC tumours demonstrate increased expression of a key collagenase, MT1-MMP [membrane-type 1 MMP (matrix metalloproteinase); also known as MMP-14]. Furthermore, a cytokine known to mediate fibrosis in vivo, TGF-β1 (transforming growth factor-β1), is up-regulated in human PDAC tumours and can promote MT1-MMP expression. In the present review, we examine the regulation of PDAC progression through the interplay between type I collagen (the most common extracellular matrix present in human PDAC tumours), MT1-MMP and TGF-β1. Specifically, we examine the way in which signalling events through these pathways mediates invasion, regulates microRNAs and contributes to chemoresistance.

Evaluating the supportive care costs of severe radiochemotherapy-lnduced mucositis and pharyngitis results from a northwestern university costs of cancer program pilot study with head and neck and nonsmall cell lung cancer patients who received care at a county hospital, a veterans administration hospital, or a comprehensive cancer care center

Few studies have examined the costs of supportive care for radiochemotherapy‐induced mucosits/pharyngitis among patients with head and neck cancer (HNC) or lung cancers despite the documented negative clinical impact of these complications.

Three dimensional collagen I promotes gemcitabine resistance in pancreatic cancer through MT1-MMP-mediated expression of HMGA2

One of the hallmarks of human pancreatic ductal adenocarcinoma (PDAC) is its pronounced type I collagen-rich fibrotic reaction. Although recent reports have shown that the fibrotic reaction can limit the efficacy of gemcitabine chemotherapy, the underlying mechanisms remain poorly understood. In this article, we show that the type I collagen allows PDAC cells to override checkpoint arrest induced by gemcitabine. Relative to cells grown on tissue culture plastic, PDAC cells grown in 3-dimensional collagen microenvironment have minimal Chk1 phosphorylation and continue to proliferate in the presence of gemcitabine. Collagen increases membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent ERK1/2 phosphorylation to limit the effect of gemcitabine. Collagen also increases MT1-MMP-dependent high mobility group A2 (HMGA2) expression, a nonhistone DNA-binding nuclear protein involved in chromatin remodeling and gene transcription, to attenuate the effect of gemcitabine. Overexpression of MT1-MMP in the collagen microenvironment increases ERK1/2 phosphorylation and HMGA2 expression, and thereby further attenuates gemcitabine-induced checkpoint arrest. MT1-MMP also allows PDAC cells to continue to proliferate in the presence of gemcitabine in a xenograft mouse model. Clinically, human tumors with increased MT1-MMP show increased HMGA2 expression. Overall, our data show that collagen upregulation of MT1-MMP contributes to gemcitabine resistance in vitro and in a xenograft mouse model, and suggest that targeting MT1-MMP could be a novel approach to sensitize pancreatic tumors to gemcitabine.

Pancreatic Cancer Cells Respond to Type I Collagen by Inducing Snail Expression to Promote Membrane Type 1 Matrix Metalloproteinase-dependent Collagen Invasion

Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Although type I collagen functions as a barrier to invasion, pancreatic cancer cells have been shown to respond to type I collagen by becoming more motile and invasive. Because epithelial-mesenchymal transition is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well known regulator of epithelial-mesenchymal transition. Relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, we showed that Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was not involved in collagen-mediated Snail expression. Overexpression of Snail in PDAC cells resulted in a robust membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14)-dependent invasion through collagen-coated transwell chambers. Snail-expressing PDAC cells also demonstrated MT1-MMP-dependent scattering in three-dimensional collagen gels. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling, and inhibiting ERK signaling in Snail-expressing cells blocked two-dimensional collagen invasion and attenuated scattering in three-dimensional collagen. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that pancreatic cancer cells increase Snail on encountering collagen-rich milieu and suggest that the desmoplastic reaction actively contributes to PDAC progression.

Contribution of Epithelial-to-Mesenchymal Transition and Cancer Stem Cells to Pancreatic Cancer Progression

Pancreatic adenocarcinoma remains among the most lethal of human malignancies. Overall 5-y survival is less than 5%, and only 20% of patients presenting with localized disease amenable to surgical resection. Even in patients who undergo resection, long-term survival remains extremely poor. A major contributor to the aggressiveness of multiple cancers, and pancreatic cancer in particular, is the process of epithelial-to-mesenchymal transition (EMT). This review highlights the growing evidence of EMT in pancreatic cancer progression, focusing on the contribution of EMT to the development of cancer stem cells and on interaction of EMT with other pathways central to cancer progression, such as Hedgehog signaling, the K-ras oncogene, and transforming growth factor-beta (TGF-β). We will also discuss EMT-targeting agents currently in development and in clinical trials that may help to reduce the morbidity and mortality associated with pancreatic cancer.

Ovarian Cancer Cell Detachment and Multicellular Aggregate Formation Are Regulated by Membrane Type 1 Matrix Metalloproteinase: A Potential Role in I.p. Metastatic Dissemination

An early event in the metastasis of epithelial ovarian carcinoma is shedding of cells from the primary tumor into the peritoneal cavity followed by diffuse i.p. seeding of secondary lesions. Anchorage-independent metastatic cells are present as both single cells and multicellular aggregates (MCA), the latter of which adhere to and disaggregate on human mesothelial cell monolayers, subsequently forming invasive foci. Although this unique metastatic mechanism presents a distinct set of therapeutic challenges, factors that regulate MCA formation and dissemination have not been extensively evaluated. Proteolytic activity is important at multiple stages in i.p. metastasis, catalyzing migration through the mesothelial monolayer and invasion of the collagen-rich submesothelial matrix to anchor secondary lesions, and acquisition of membrane type 1 matrix metalloproteinase (MT1-MMP; MMP-14) expression promotes a collagen-invasive phenotype in ovarian carcinoma. MT1-MMP is regulated posttranslationally through multiple mechanisms including phosphorylation of its cytoplasmic tail, and the current data using ovarian cancer cells expressing wild-type, phosphomimetic (T567E-MT1-MMP), and phosphodefective (T567A-MT1-MMP) MT1-MMP show that MT1-MMP promotes MCA formation. Confluent T567E-MT1-MMP-expressing cells exhibit rapid detachment kinetics, spontaneous release as cell-cell adherent sheets concomitant with MT1-MMP-catalyzed alpha(3) integrin ectodomain shedding, and robust MCA formation. Expansive growth within three-dimensional collagen gels is also MT1-MMP dependent, with T567E-MT1-MMP-expressing cells exhibiting multiple collagen invasive foci. Analysis of human ovarian tumors shows elevated MT1-MMP in metastases relative to paired primary tumors. These data suggest that MT1-MMP activity may be key to ovarian carcinoma metastatic success by promoting both formation and dissemination of MCAs.

Slug is a downstream mediator of transforming growth factor‐β1‐induced matrix metalloproteinase‐9 expression and invasion of oral cancer cells

Members of Snail family of transcription factors play an important role in oral cancer progression by inducing epithelial–mesenchymal transition, by promoting invasion and by increasing matrix metalloproteinase (MMP) expression. Although Snail (Snai1) is the best characterized and the most extensively studied member of this family, the role and regulation of Slug (Snai2) in oral cancer progression is less well understood. In this report, we show that transforming growth factor‐β1 (TGF‐β1) increases Slug levels in tert‐immortalized oral keratinocytes and in malignant oral squamous cell carcinoma (OSCC) cells. Inhibiting ERK1/2 signaling, but not PI3‐kinase signaling, blocked TGF‐β1‐induced Slug expression in the malignant UMSCC1 cells. To further examine the role of Slug in OSCC progression, we generated UMSCC1 cells with inducible expression of Slug protein. Induction of Slug in UMSCC1 cells did not repress E‐cadherin levels or regulate individual movement of UMSCC1 cells. Instead, Slug enhanced cohort migration and Matrigel invasion by UMSCC1 cells. Slug increased MMP‐9 levels and MMP‐9‐specific siRNA blocked Slug‐induced Matrigel invasion. Interestingly, Slug‐specific siRNA attenuated TGF‐β1‐induced MMP‐9 expression and Matrigel invasion. These data demonstrate that TGF‐β1 increases Slug via ERK1/2 signaling, and thereby contributes to OSCC progression. J. Cell. Biochem. 108: 726–736, 2009.

Mast cell 5-lipoxygenase activity promotes intestinal polyposis in APC Δ468 mice

Arachidonic acid metabolism has been implicated in colon carcinogenesis, but the role of hematopoietic 5-lipoxygenase (5LO) that may impact tumor immunity in development of colon cancer has not been explored. Here we show that tissue-specific deletion of the 5LO gene in hematopoietic cells profoundly attenuates polyp development in the APC(Δ468) murine model of colon polyposis. In vitro analyses indicated that mast cells in particular utilized 5LO to limit proliferation of intestinal epithelial cells and to mobilize myeloid-derived suppressor cells (MDSCs). Mice lacking hemapoietic expression of 5LO exhibited reduced recruitment of MDSCs to the spleen, mesenteric lymph nodes, and primary tumor site. 5LO deficiency also reduced the activity in MDSCs of arginase-1, which is thought to be critical for MDSC function. Together, our results establish a pro-tumorigenic role of hematopoietic 5LO in the immune microenvironment and suggest 5LO inhibition as an avenue for future investigation in treatment of colorectal polyposis and cancer.