Mario A. Shields

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.

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.

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.

Interplay between β1-Integrin and Rho Signaling Regulates Differential Scattering and Motility of Pancreatic Cancer Cells by Snail and Slug Proteins

Background: This study was designed to compare the role of Snail and Slug in pancreatic cancer. Results: Snail and Slug have differential effects on three-dimensional scattering, motility, adhesion, and Rho signaling. Conclusion: Snail, but not Slug, promotes motility and scattering in three-dimensional collagen of pancreatic cancer cells. Significance: Understanding how Snail and Slug modulate pancreatic cancer progression may identify therapeutic targets for this deadly disease. The Snail family of transcription factors has been implicated in pancreatic cancer progression. We recently showed that Snail (Snai1) promotes membrane-type 1 matrix metalloproteinase (MT1-MMP)- and ERK1/2-dependent scattering of pancreatic cancer cells in three-dimensional type I collagen. In this study, we examine the role of Slug (Snai2) in regulating pancreatic cancer cell scattering in three-dimensional type I collagen. Although Slug increased MT1-MMP expression and ERK1/2 activity, Slug-expressing cells failed to scatter in three-dimensional collagen. Moreover, in contrast to Snail-expressing cells, Slug-expressing cells did not demonstrate increased collagen I binding, collagen I-driven motility, or α2β1-integrin expression. Significantly, inhibiting β1-integrin function decreased migration and scattering of Snail-expressing cells in three-dimensional collagen. As Rho GTPases have been implicated in invasion and migration, we also analyzed the contribution of Rac1 and Rho signaling to the differential migration and scattering of pancreatic cancer cells. Snail-induced migration and scattering were attenuated by Rac1 inhibition. In contrast, inhibiting Rho-associated kinase ROCK1/2 increased migration and scattering of Slug-expressing cells in three-dimensional collagen and thus phenocopied the effects of Snail in pancreatic cancer cells. Additionally, the increased migration and scattering in three-dimensional collagen of Slug-expressing cells following ROCK1/2 inhibition was dependent on β1-integrin function. Overall, these results demonstrate differential effects of Snail and Slug in pancreatic cancer and identify the interplay between Rho signaling and β1-integrin that functions to regulate the differential scattering and migration of Snail- and Slug-expressing pancreatic cancer cells.

Role of Interferon α (IFNα)-inducible Schlafen-5 in Regulation of Anchorage-independent Growth and Invasion of Malignant Melanoma Cells

IFNα exerts potent inhibitory activities against malignant melanoma cells in vitro and in vivo, but the mechanisms by which it generates its antitumor effects remain unknown. We examined the effects of interferon α (IFNα) on the expression of human members of the Schlafen (SLFN) family of genes, a group of cell cycle regulators that mediate growth-inhibitory responses. Using quantitative RT-real time PCR, we found detectable basal expression of all the different human SLFN genes examined (SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14), in malignant melanoma cells and primary normal human melanocytes, but SLFN5 basal expression was suppressed in all analyzed melanoma cell lines. Treatment of melanoma cells with IFNα resulted in induction of expression of SLFN5 in malignant cells, suggesting a potential involvement of this gene in the antitumor effects of IFNα. Importantly, stable knockdown of SLFN5 in malignant melanoma cells resulted in increased anchorage-independent growth, as evidenced by enhanced colony formation in soft agar assays. Moreover, SLFN5 knockdown also resulted in increased invasion in three-dimensional collagen, suggesting a dual role for SLFN5 in the regulation of invasion and anchorage-independent growth of melanoma cells. Altogether, our findings suggest an important role for the SLFN family of proteins in the generation of the anti-melanoma effects of IFNα and for the first time directly implicate a member of the human SLFN family in the regulation of cell invasion.

MT1-MMP cooperates with Kras(G12D) to promote pancreatic fibrosis through increased TGF-β signaling.

Pancreatic cancer is associated with a pronounced fibrotic reaction that was recently shown to limit delivery of chemotherapy. To identify potential therapeutic targets to overcome this fibrosis, we examined the interplay between fibrosis and the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14), which is required for growth and invasion in the collagen-rich microenvironment. In this article, we show that compared with control mice (Kras+/MT1-MMP−) that express an activating KrasG12D mutation necessary for pancreatic cancer development, littermate mice that express both MT1-MMP and KrasG12D (Kras+/MT1-MMP+) developed a greater number of large, dysplastic mucin-containing papillary lesions. These lesions were associated with a significant amount of surrounding fibrosis, increased α-smooth muscle actin (+) cells in the stroma, indicative of activated myofibroblasts, and increased Smad2 phosphorylation. To further understand how MT1-MMP promotes fibrosis, we established an in vitro model to examine the effect of expressing MT1-MMP in pancreatic ductal adenocarcinoma (PDAC) cells on stellate cell collagen deposition. Conditioned media from MT1-MMP–expressing PDAC cells grown in three-dimensional collagen enhanced Smad2 nuclear translocation, promoted Smad2 phosphorylation, and increased collagen production by stellate cells. Inhibiting the activity or expression of the TGF-β type I receptor in stellate cells attenuated MT1-MMP conditioned medium–induced collagen expression by stellate cells. In addition, a function-blocking anti–TGF-β antibody also inhibited MT1-MMP conditioned medium–induced collagen expression in stellate cells. Overall, we show that the bona fide collagenase MT1-MMP paradoxically contributes to fibrosis by increasing TGF-β signaling and that targeting MT1-MMP may thus help to mitigate fibrosis. Mol Cancer Res; 9(10); 1294–304. ©2011 AACR.

Snail cooperates with KrasG12D to promote pancreatic fibrosis

Patients with pancreatic cancer, which is characterized by an extensive collagen-rich fibrotic reaction, often present with metastases. A critical step in cancer metastasis is epithelial-to-mesenchymal transition (EMT), which can be orchestrated by the Snail family of transcription factors. To understand the role of Snail (SNAI1) in pancreatic cancer development, we generated transgenic mice expressing Snail in the pancreas. Because chronic pancreatitis can contribute to pancreatic cancer development, Snail-expressing mice were treated with cerulein to induce pancreatitis. Although significant tissue injury was observed, a minimal difference in pancreatitis was seen between control and Snail-expressing mice. However, because Kras mutation is necessary for tumor development in mouse models of pancreatic cancer, we generated mice expressing both mutant KrasG12D and Snail (Kras+/Snail+). Compared with control mice (Kras+/Snai−), Kras+/Snail+ mice developed acinar ectasia and more advanced acinar-to-ductal metaplasia. The Kras+/Snail+ mice exhibited increased fibrosis, increased phosphorylated Smad2, increased TGF-β2 expression, and activation of pancreatic stellate cells. To further understand the mechanism by which Snail promoted fibrosis, we established an in vitro model to examine the effect of Snail expression in pancreatic cancer cells on stellate cell collagen production. Snail expression in pancreatic cancer cells increased TGF-β2 levels, and conditioned media from Snail-expressing pancreatic cancer cells increased collagen production by stellate cells. Additionally, inhibiting TGF-β signaling in stellate cells attenuated the conditioned media–induced collagen production by stellate cells. Together, these results suggest that Snail contributes to pancreatic tumor development by promoting fibrotic reaction through increased TGF-β signaling. Implications: Expression of the EMT regulator Snail in the context of mutant Kras provides new insight into pancreatic cancer progression. Mol Cancer Res; 11(9); 1078–87. ©2013 AACR.

Rho-ROCK-Myosin Signaling Meditates Membrane Type 1 Matrix Metalloproteinase-induced Cellular Aggregation of Keratinocytes

Membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), which is associated with extracellular matrix (ECM) breakdown in squamous cell carcinoma (SCC), promotes tumor formation and epithelial-mesenchymal transition. However, in this report we demonstrate that MT1-MMP, by cleaving the underlying ECM, causes cellular aggregation of keratinocytes and SCC cells. Treatment with an MMP inhibitor abrogated MT1-MMP-induced phenotypic changes, but decreasing E-cadherin expression did not affect MT1-MMP-induced cellular aggregation. As ROCK1/2 can regulate cell-cell and cell-ECM interaction, we examined its role in mediating MT1-MMP-induced phenotypic changes. Blocking ROCK1/2 expression or activity abrogated the cellular aggregation resulting from MT1-MMP expression. Additionally, blocking Rho and non-muscle myosin attenuated MT1-MMP-induced phenotypic changes. Moreover, SCC cells expressing only the catalytically active MT1-MMP protein demonstrated increased cellular aggregation and increased myosin II activity in vivo when injected subcutaneously into nude mice. Together, these results demonstrate that expression of MT1-MMP may be anti-tumorigenic in keratinocytes by promoting cellular aggregation.