Collagen regulation of let-7 in pancreatic cancer involves TGF-β1-mediated membrane type 1-matrix metalloproteinase expression

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced collagen-rich fibrosis known as desmoplastic reaction; however, the role of fibrosis in PDAC is poorly understood. In this report we show that collagen can regulate the tumor suppressive let-7 family of microRNAs in pancreatic cancer cells. PDAC cells growing in 3D collagen gels repress mature let-7 without affecting the precursor form of let-7 in part through increased expression of membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) and ERK1/2 activation. PDAC cells in collagen also demonstrate increased TGF-β1 signaling, and blocking TGF-β1 signaling attenuated collagen-induced MT1-MMP expression, ERK1/2 activation and repression of let-7 levels. Although MT1-MMP overexpression was not sufficient to inhibit let-7 on 2D tissue culture plastic, overexpression of MT1-MMP in PDAC cells embedded in 3D collagen gels or grown in vivo repressed let-7 levels. Importantly, MT1-MMP expression significantly correlated with decreased levels of let-7 in human PDAC tumor specimens. Overall, our study emphasizes the interplay between the key proteinase MT1-MMP and its substrate type I collagen in modulating microRNA expression, and identifies an additional mechanism by which fibrosis may contribute to PDAC progression.     

Extracellular matrix-mediated membrane-type 1 matrix metalloproteinase expression in pancreatic ductal cells is regulated by transforming growth factor-beta1

Pancreatic ductal adenocarcinoma (PDAC) is associated with an intense fibrotic reaction around the tumor known as desmoplastic reaction. This tissue is composed of interstitial matrix, predominantly type I collagen, together with proliferating fibroblastic cells. Despite the recognized importance of tumor-stromal interactions, very little is known about the interactions among pancreatic cells, myofibroblasts, and the interstitial matrix. The current study was undertaken to test the hypothesis that the desmoplastic reaction alters PDAC gene expression and cellular behavior. Evaluation of human pancreatic specimens showed increased fibrosis and enhanced membrane type 1-matrix metalloproteinase (MT1-MMP) expression in tumor specimens compared with normal pancreas. Using an in vitro model of tumor cell-stromal interactions, type I collagen and the extracellular matrix deposited by pancreatic fibroblasts and PDAC cells regulated motility of human papillomavirus-immortalized human pancreatic ductal epithelial (HPDE) cells. These "stromal" matrices also regulated MT1-MMP expression by HPDE cells, without affecting the expression of tissue inhibitor of metalloproteinase 2. Treatment with transforming growth factor-beta1 (TGF-beta1) type I receptor kinase inhibitors and function-blocking anti-TGF-beta1 antibody abrogated matrix-mediated MT1-MMP induction. TGF-beta1 also promoted MT1-MMP-dependent migration by HPDE cells. Moreover, compared with normal tissue, there was increased TGF-beta1 signaling in grade 3 tumor specimens as shown by increased phospho-Smad2 staining. These data show that the crosstalk between cancer cells and stromal elements mediated by TGF-beta1 influences cell surface- and pericellular matrix-degrading potential in vitro and may contribute to pancreatic cancer progression in vivo.     

Impaired tyrosine phosphorylation of membrane type 1-matrix metalloproteinase reduces tumor cell proliferation in three-dimensional matrices and abrogates tumor growth in mice

Pericellular proteolysis of the extracellular matrix by membrane type 1-matrix metalloproteinase (MT1-MMP) confers tumor cells with the ability to proliferate within three-dimensional (3D) matrices and sustains tumor growth in mice. In this study, we show that in addition to its matrix-degrading activity, phosphorylation of MT1-MMP on its unique tyrosine residue located within its cytoplasmic sequence (Tyr573) may also participate to these processes. Fibrosarcoma cells expressing a proteolytically active but non-phosphorylable mutant of MT1-MMP showed a markedly reduced proliferation rate when embedded within 3D type I collagen matrices, this antiproliferative effect being correlated with arrest in the G(0)/G(1) phase of the cell cycle. Impaired tyrosine phosphorylation of MT1-MMP also inhibits anchorage-independent growth of HT-1080 cells in soft agar as well as their invasion of collagen barriers, two prominent attributes of tumor cells, suggesting a broad inhibitory effect of the MT1-MMP mutant on tumorigenesis. Accordingly, whereas HT-1080 cells formed well-vascularized tumors containing tyrosine-phosphorylated MT1-MMP, tumor growth was completely abolished by expression of the non-phosphorylable MT1-MMP mutant. These findings thus indicate a close co-operation between the matrix-degrading activity of MT1-MMP and tyrosine phosphorylation of its intracellular domain for tumor cell invasion and proliferation and suggest that the targeting of the intracellular signaling pathways leading to tyrosine phosphorylation of MT1-MMP may represent an unexpected alternative strategy for the inhibition of this enzyme.     

Membrane type 1 matrix metalloproteinase regulates collagen-dependent mitogen-activated protein/extracellular signal-related kinase activation and cell migration

Mitogen-activated protein kinase-extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signaling has been implicated in the regulation of tumor cell invasion and metastasis. Migration of HT1080 cells on type I collagen was suppressed by the matrix metalloproteinase (MMP) inhibitors BB94 and tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1. TIMP-2-specific inhibition suggests that membrane type 1 MMP (MT1-MMP) is likely involved in this process. Activation of ERK was induced in HT1080 cells adhered on dishes coated with type I collagen, and this was inhibited by BB94. MMP-2 processing in HT1080 cells, which also was stimulated by cultivation on type I collagen, was inhibited by MEK inhibitor PD98059. Expression of a constitutively active form of MEK1 promoted MMP-2 processing concomitant with the increase of MT1-MMP levels, suggesting that MT1-MMP is regulated by MEK/ERK signaling. In addition, expression of the hemopexin-like domain of MT1-MMP in HT1080 cells interfered with MMP-2 processing, ERK activation, and cell migration, implying that the enzymatic activity of MT1-MMP is involved in collagen-induced ERK activation, which results in enhanced cell migration. Thus, adhesion of HT1080 cells to type I collagen induces MT1-MMP-dependent ERK activation, which in turn causes an increase in MT1-MMP levels and subsequent cell migration.     

HMGA-targeted phosphorothioate DNA aptamers increase sensitivity to gemcitabine chemotherapy in human pancreatic cancer cell lines

Elevated high mobility group A (HMGA) protein expression in pancreatic cancer cells is correlated with resistance to the chemotherapy agent gemcitabine. Here, we demonstrate use of HMGA-targeted AT-rich phosphorothioate DNA (AT-sDNA) aptamers to suppress HMGA carcinogenic activity. Cell growth of human pancreatic cancer cells (AsPC-1 and Miapaca-2) transfected with AT-sDNA were monitored after treatment with gemcitabine. Significant increases in cell death in AT-sDNA transfected cells compared to non-AT-rich sDNA treated cells were observed in both cell lines. The data indicate the potential use of HMGA targeted DNA aptamers to enhance chemotherapy efficacy in pancreatic cancer treatment.     

Novel MT1-MMP small-molecule inhibitors based on insights into hemopexin domain function in tumor growth

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a promising drug target in malignancy. The structure of MT1-MMP includes the hemopexin domain (PEX) that is distinct from and additional to the catalytic domain. Current MMP inhibitors target the conserved active site in the catalytic domain and, as a result, repress the proteolytic activity of multiple MMPs instead of MT1-MMP alone. In our search for noncatalytic inhibitors of MT1-MMP, we compared the protumorigenic activity of wild-type MT1-MMP with an MT1-MMP mutant lacking PEX (ΔPEX). In contrast to MT1-MMP, ΔPEX did not support tumor growth in vivo, and its expression resulted in small fibrotic tumors that contained increased levels of collagen. Because these findings suggested an important role for PEX in tumor growth, we carried out an inhibitor screen to identify small molecules targeting the PEX domain of MT1-MMP. Using the Developmental Therapeutics Program (National Cancer Institute/NIH), virtual ligand screening compound library as a source and the X-ray crystal structure of PEX as a target, we identified and validated a novel PEX inhibitor. Low dosage, intratumoral injections of PEX inhibitor repressed tumor growth and caused a fibrotic, ΔPEX-like tumor phenotype in vivo. Together, our findings provide a preclinical proof of principle rationale for the development of novel and selective MT1-MMP inhibitors that specifically target the PEX domain.     

Expression of membrane type 1 matrix metalloproteinase is associated with cervical carcinoma progression and invasion

Membrane type 1 matrix metalloproteinase (MT1-MMP) is frequently expressed by cancer cells and is believed to play an important role in cancer cell invasion and metastasis. However, little is known about the role of MT1-MMP in mediating invasiveness of cervical cancer cells. In this study, we examined MT1-MMP expression in 58 primary human cervical tissue specimens, including normal cervix, low-grade squamous intraepithelial lesions (LSIL), high-grade SILs (HSIL), and invasive carcinomas. We also evaluated MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2 expression in several cervical cancer-derived cell lines, human papillomavirus (HPV)-immortalized keratinocytes, and keratinocytes derived from a LSIL. Using in situ hybridization techniques to study the cervical tissue specimens, we found that MT1-MMP expression increases with cervical tumor progression (Spearman correlation coefficient = 0.66; P < 0.0001, exact test). Specifically, MT1-MMP expression is very low or absent in normal cervix and LSILs, is readily detectable in HSILs, and is very strongly expressed in nearly all invasive carcinomas. Most but not all cervical cancer-derived cell lines also expressed significant levels of MT1-MMP and MMP-2. Constitutive expression of exogenous MT1-MMP in cervical carcinoma-derived cells and HPV-immortalized keratinocytes with low endogenous levels of MT1-MMP induced invasiveness in collagen I, but this effect was not observed in LSIL-derived keratinocytes. Our results show that MT1-MMP is a key enzyme mediating cervical cancer progression. However, MT1-MMP alone is not always sufficient for inducing keratinocyte invasiveness at least in the collagen I invasion assay used in this study. Further studies of gene expression in preinvasive and invasive cervical cancers should assist with identification of additional critical factors mediating cervical cancer progression.     

The growth inhibition of hepatocellular and cholangiocellular carcinoma cells by gemcitabine and the roles of extracellular signal-regulated and checkpoint kinases

We examined the effects of gemcitabine, a pyrimidine analogue, on hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) cells. After HCC cells (HepG2, Hep3B, HLF and PLC/PRF/5) and CCC cells (HuCCT-1) were treated with gemcitabine, cellular growth, cell cycle, nuclear morphology and activity of signaling molecules were evaluated by WST-8 assays, flow cytometry analysis, Hoechst 33258 staining and Western blotting, respectively. We found that gemcitabine significantly inhibited the growth of HCC and CCC cells in a dose- and time-dependent manner. Gemcitabine induced cell cycle arrest at the G1 phase, however, the sub-G1 fraction was not observed and nuclear morphology did not indicate the induction of apoptosis. Gemcitabine induced differential activation of checkpoint kinases, Chk2 and Chk1, in HCC and CCC cells, respectively and gemcitabine activated extracellular signal-regulated kinase (ERK)1/2 in both cell types. After the cells were pretreated with a MEK inhibitor U0126, activations of these checkpoint kinases were abrogated and the cell death was enhanced. These results demonstrate that gemcitabine inhibited the growth of HCC and CCC cells by cell cycle arrest without apoptosis and that the ERK/Chk1/2 signaling pathway was in part responsible for the resistance to gemcitabine. Our findings shed light on treating patients with HCC and CCC by gemcitabine, especially when combined with a MEK inhibitor and Chk1/2 inhibitors.     

膜型基质金属蛋白酶-1在基质金属蛋白酶-2活化中的作用机制

目的:研究MMP-2的活化并探索其机制.方法:采用酶谱分析方法检测瘤细胞条件培养基中MMP-2在包被前后表达和活化的情况,用RT-PCR方法检测MT1-MMP mRNA的表达,观察MT1-MMP激活的MMP-2在细胞侵袭中的作用,Boyden小室膜侵袭实验检测细胞的侵袭能力.结果:条件培养基明胶酶谱分析结果显示仅在三维聚I型胶原包被组存在MMP-2的活性形式;培养于I型胶原组的人MCF-7细胞MT1-MMP mRNA表达量明显高于对照组,与对照组间差异显著(P<0.01).Boyden小室膜侵袭实验可见,用MT1-MMP的抗体处理组的细胞数明显少于未用MT1-MMP的抗体处理组,差异有统计学意义(P<0.01).结论:I型胶原成分可以通过上调MT1-MMP的水平来调控人乳腺癌MCF-7细胞MMP-2的活化,MT1-MMP激活的MMP-2可以增强人乳腺癌MCF-7细胞的侵袭能力.     

Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type 1-MMP-dependent tumor growth in mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.     

EGF调节MT1-MMP和MMP-2的表达及其信号传导机制的研究

目的:探讨表皮生长因子（EGF）在宫颈癌SiHa细胞中对膜型基质金属蛋白酶（MT1-MMP）和基质金属蛋白酶-2（MMP-2）表达的调节作用,明确其相关的信号传导机制。方法:利用EGF作为干预因素,信号通路阻断剂分别阻断表皮生长因子受体（EGFR）、AKT、ERK、p38和JNK的磷酸化,来观察EGF对MT1-MMP和MMP-2表达的影响及相关信号通路。结果:EGF在mRNA和蛋白水平上诱导MT1-MMP的表达增加和抑制MMP-2的表达;MAPK和ERK激酶抑制剂在mRNA和蛋白水平上可阻断这种诱导;PI3-K抑制剂不影响EGF对MT1-MMP的诱导,但可进一步抑制MMP-2的表达。结论:EGFR通过MAPK/ERK信号通路上调MP1-MMP的表达和下调MMP-2的表达,同时还通过PI3-K/AKT的信号通路轻度上调MMP-2的表达。此外,EGF可以提高细胞培养基中MMP-2的活性。     

Therapeutic significance of elevated tissue transglutaminase expression in pancreatic cancer.

PURPOSE: Tissue transglutaminase (TG2) is a multifunctional protein that is implicated in development of drug resistance and metastasis. Therefore, we examined therapeutic targeting of TG2 for inhibiting growth and metastasis of in vivo growing pancreatic ductal adenocarcinoma (PDAC) in nude mice. EXPERIMENTAL DESIGN: We implanted Panc-28 pancreatic cancer cells to induce orthotopic PDAC tumors in nude mice and determined the efficacy of liposomal TG2 small interfering RNA (siRNA) either alone or in combination with gemcitabine. RESULTS: We show that down-regulation of endogenous TG2 by siRNA could effectively block the growth of PDAC. Moreover, down-regulation of TG2 significantly enhanced the therapeutic efficacy of gemcitabine against PDAC and inhibited metastatic spread of the disease. The antitumor activity was related to inhibition of proliferation, angiogenesis, and Akt phosphorylation. CONCLUSION: siRNA-mediated down-regulation of TG2 represents a promising therapeutic approach for improved treatment of PDAC.     

Inhibition of membrane-type 1 matrix metalloproteinase at cell-matrix adhesions

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated in tumor invasion and metastasis. We previously reported that extracellular matrix degradation by MT1-MMP regulates cell migration via modulating sustained integrin-mediated signals. In this study, MT1-MMP-expressing cells were plated onto fibronectin-coated plates and monitored for cell-matrix adhesion formation and fibronectin degradation. The fibronectin was degraded and removed in line with the cell migration track. The migrating cells showed a polarized morphology and were in contact with the edge of fibronectin through the leading edge, in which cell-matrix adhesions are concentrated. Expression of MT1-MMP targeted to cell-matrix adhesions by fusing with the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) promoted the initial fibronectin lysis at the cell periphery immediately after adhesion. These results suggest that fibronectin is degraded by MT1-MMP located at cell-matrix adhesions, which are concentrated at the leading edge of the migrating cells. To inhibit MT1-MMP at cell-matrix adhesion, the dominant negative form of MT1-MMP (MT1-Pex) was targeted to the cell-matrix adhesion by fusing with the FAT domain (MT1-Pex-FAT). MT1-Pex-FAT accumulated at cell-matrix adhesions and inhibited fibronectin degradation as well as FAK phosphorylation more effectively than parental MT1-Pex. MT1-Pex-FAT was also shown to suppress the invasion of tumor cells into three-dimensional collagen gel more strongly than MT1-Pex. These results suggest that MT1-MMP-mediated extracellular matrix lysis at cell-matrix adhesions induces the establishment of cell polarity, which facilitates cell-matrix adhesion turnover and subsequent cell migration. This model highlights the role of MT1-MMP at the leading edge of migrating cells.     

Inhibition of oncogenic Pim-3 kinase modulates transformed growth and chemosensitizes pancreatic cancer cells to gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival rate of only 6%. Although the cytosine analog gemcitabine is the drug commonly used to treat PDAC, chemoresistance unfortunately renders the drug ineffective. Thus, strategies that can decrease this resistance will be essential for improving the dismal outcome of patients suffering from this disease. We previously observed that oncogenic Pim-1 kinase was aberrantly expressed in PDAC tissues and cell lines and was responsible for radioresistance. Furthermore, members of the Pim family have been shown to reduce the efficacy of chemotherapeutic drugs in cancer. Therefore, we attempted to evaluate the role of Pim-3 in chemoresistance of PDAC cells. We were able to confirm upregulation of the Pim-3 oncogene in PDAC tissues and cell lines vs. normal samples. Biological consequences of inhibiting Pim-3 expression with shRNA-mediated suppression included decreases in anchorage-dependent growth, invasion through Matrigel and chemoresistance to gemcitabine as measured by caspase-3 activity. Additionally, we were able to demonstrate that Pim-1 and Pim-3 play overlapping but non-identical roles as it relates to gemcitabine sensitivity of pancreatic cancer cells. To further support the role of Pim-3 suppression in sensitizing PDAC cells to gemcitabine, we used the pharmacological Pim kinase inhibitor SGI-1776. Treatment of PDAC cells with SGI-1776 resulted in decreased phosphorylation of the proapoptotic protein Bad and cell cycle changes. When SGI-1776 was combined with gemcitabine, there was a greater decrease in cell viability in the PDAC cells vs. cells treated with either of the drugs separately. These results suggest combining drug therapies that inhibit Pim kinases, such as Pim-3, with chemotherapeutic agents, to aid in decreasing chemoresistance in pancreatic cancer.     

Irradiation-induced G2/M checkpoint response requires ERK1/2 activation

Following DNA damage, cells undergo G2/M cell cycle arrest, allowing time for DNA repair. G2/M checkpoint activation involves activation of Wee1 and Chk1 kinases and inhibition of Cdc25A and Cdc25C phosphatases, which results in inhibition of Cdc2 kinase. Results presented in this report indicate that gamma-irradiation (IR) exposure of MCF-7 cells resulted in extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation and induction of G2/M arrest. Furthermore, inhibition of ERK1/2 signaling resulted in >or=85% attenuation in IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ataxia telangiectasia mutated- and rad3-related (ATR), Chk1 and Wee1 kinases as well as phosphorylation of Cdc25A-Thr506, Cdc25C-Ser216 and Cdc2-Tyr15. Moreover, incubation of cells with caffeine, which inhibits ataxia telangiectasia mutated (ATM)/ATR, or transfection of cells with short interfering RNA targeting ATR abrogated IR-induced Chk1 phosphorylation and G2/M arrest but had no effect on IR-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling resulted in marked attenuation in IR-induced ATR activity with little, if any, effect on IR-induced ATM activation. These results implicate IR-induced ERK1/2 activation as an important regulator of G2/M checkpoint response to IR in MCF-7 cells.