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Effects of hepatocyte growth factor on the expression of matrix metalloproteinases and their tissue inhibitors during the endometrial cancer invasion in a three‐dimensional coculture
Abstract
Matrix metalloproteinase (MMP)-2 and -9 are secreted and translocated from endometrial stromal cells to HEC-1 A cells in a steroid-dependent manner. We investigated the paracrine effect of hepatocyte growth factor (HGF) on MMPs and metalloproteinase tissue inhibitor (TIMP) expression in stromal and endometrial cancer cells, and correlated with cancer cell invasiveness in three-dimensional (3D) coculture. The 3D coculture of endometrial stromal and cancer cell lines (HEC-1 A, HEC-IB, or KLE) were maintained in the presence or absence of HGF. The expression of MMP-2 and -9, MT1-MMP, TIMP-1 and -2 were examined by RT-PCR and zymography. Under the same conditions, invasion of the cancer cells was quantified by Boyden's chamber assay. HGF strongly induced MMP-9 mRNA expression in stromal cells, but had little effect on MMP-2 mRNA. MT1-MMP mRNA was detected only in KLE and stromal cells, which was also increased by HGF. TIMP-1 and -2 mRNAs was ubiquitous with no dependence on HGF. Zymographic analysis of MMPs showed that activation of MMP-2 and -9 was enhanced by HGF. A significant increase in invasion of all three cancer cells with HGF was observed. The effect of HGF on the invasiveness of 3D cocultured endometrial cancer cells and stromal cells appears to be due to induction of MMP-9 mRNA expression in stromal cells and /or increased activation of MMP-2 and MMP-9 by proteolytic digestion.
... 21 Yet, questions remain to be answered as to what directs the invasion of the cancer under the estrogen influence. In view of HGF as a key element inducing the endometrial cancer invasion, 22,23 we attempt to elucidate (i) whether HGF correlates with the invasion of endometrial cancer cells in response to varying ovarian hormones, and herein, (ii) to what extent HGF plays a role in mediating the influence of ovarian hormones. ...
... Human endometrial stromal cells were isolated as previously described. 21,22 In brief, human endometrial tissue was isolated by curettage in hysterectomy from the uteri of patients who underwent surgery at Ajou University Hospital (Suwon, South Korea), but showed no sign of endometrial diseases. Informed consent was obtained from the patients before surgery and IRB approval was granted from the Ajou University Hospital. ...
... Many previous studies support our results in that endometrial cancer cells are sensitive to HGF for increased invasion and motility, [22][23][24] and stromal cells are the cells that secret HGF. [5][6][7][8][9][10][11] Moreover, 2 estrogen response elements were found in the 5 0 upstream regions of the mouse HGF gene, suggesting transcriptional regulation by estrogen. ...
Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor-alpha expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF-antagonist/angiogenesis inhibitor)-sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin alpha(v)beta(5) as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention.
... Besides MMP-7, MMP-2 is secreted in relative low amounts in HEC1A conditioned medium. While primary endometrial cancer cell cultures have been found to express MMP-2, the literature conflicts as to whether HEC1A cells produce endogenous MMP-2 [42][43][44][45][46]. By gelatin zymography, we detected low levels of proMMP-2 secretion in the concentrated conditioned medium that was collected from invading HEC1A cells. ...
... The literature is conflicted as to whether endometrial cancer cells in general, and HEC1A cells in particular, produce endogenous MMP-2. Some reports have found low levels of MMP-2 [42][43], and others have not [44][45][46]. It has been demonstrated that exogenous MMP-2 derived from surrounding stromal cells is translocated onto the HEC1A cell surface where its binds, in part, via the integrin β3 subunit [44]. ...
Objective:
We have previously shown that lysophosphatidic acid (LPA) promotes the ovarian cancer metastatic cascade. In this study, we evaluated the role of LPA on endometrial cancer invasion.
Methods:
Transient mRNA knockdown was accomplished using pre-designed siRNA duplexes against LPA receptor 2 (LPA2) and human matrix metalloproteinase-7 (MMP-7). RT-PCR was used to characterize LPA receptor and MMP-7 expression. Analysis of in vitro invasion was performed with rat-tail collagen type I coated Boyden chambers. Gelatin zymography was used to evaluate the MMP activity in cell culture conditioned media. Cell-cell and cell-matrix attachment was also assessed upon LPA2 knockdown to further illuminate the LPA2 cascade.
Results:
LPA increases HEC1A cellular invasion at physiologic concentrations (0.1-1 muM). Of the four principle LPA receptors, LPA2 is predominantly expressed by HEC1A cells. Transient transfection of LPA2 siRNA reduced LPA2 mRNA expression in HEC1A cells by 93% (P<0.01). Silencing LPA2 eliminated the LPA-stimulated increase in invasion (P<0.05) and reduced LPA-induced MMP-7 secretion/activation, without significantly affecting cell-cell or cell-matrix adhesion. Silencing MMP-7 reduced overall invasion but did not eliminate LPA's pro-invasive effect on HEC1A cells, as compared to negative control (P<0.05). Gelatin zymography confirmed that LPA2 and MMP-7 knockdown reduced MMP-7 activation in HEC1A conditioned media.
Conclusion:
LPA2 mediates LPA-stimulated HEC1A invasion and the subsequent activation of MMP-7.
... Cell motility and invasion experiments in our group had previously established the functional relevance of HGF-c-Met signaling as promoter of MB single cell migration on flat surfaces and inside three-dimensional collagen matrix. These observations complied with previous findings that have reported HGF induction of 3D cell motility in ductal adenocarcinoma, human mammary fibroblasts as well as endometrial carcinoma (Park, Ryu et al. 2003;Doyle, Carvajal et al. 2015;Spina, De Pasquale et al. 2015). ...
... MMP9 encodes a matrix metalloproteinase, involved in the breakdown of the extracellular matrix, a process which has been well studied for its relationship with cancer. MMP9 is secreted from endometrial stromal cells in response to induction by growth factors, such as HGF, in endometrial cancer cell lines, which, in turn, increases cancer cell invasiveness (31). Expression of MMP9 is known to be up-regulated through pro-inflammatory cytokines, including nuclear factor kappa B, IL8, and TNF-alpha, leading to increased tumor cell proliferation (32)(33)(34). ...
Background:
Experimental and epidemiologic evidence have suggested that chronic inflammation may play a critical role in endometrial carcinogenesis.
Methods:
To investigate this hypothesis, a two-stage study was carried out to evaluate single-nucleotide polymorphisms (SNP) in inflammatory pathway genes in association with endometrial cancer risk. In stage I, 64 candidate pathway genes were identified and 4,542 directly genotyped or imputed SNPs were analyzed among 832 endometrial cancer cases and 2,049 controls, using data from the Shanghai Endometrial Cancer Genetics Study. Linkage disequilibrium of stage I SNPs significantly associated with endometrial cancer (P < 0.05) indicated that the majority of associations could be linked to one of 24 distinct loci. One SNP from each of the 24 loci was then selected for follow-up genotyping. Of these, 21 SNPs were successfully designed and genotyped in stage II, which consisted of 10 additional studies including 6,604 endometrial cancer cases and 8,511 controls.
Results:
Five of the 21 SNPs had significant allelic odds ratios (ORs) and 95% confidence intervals (CI) as follows: FABP1, 0.92 (0.85-0.99); CXCL3, 1.16 (1.05-1.29); IL6, 1.08 (1.00-1.17); MSR1, 0.90 (0.82-0.98); and MMP9, 0.91 (0.87-0.97). Two of these polymorphisms were independently significant in the replication sample (rs352038 in CXCL3 and rs3918249 in MMP9). The association for the MMP9 polymorphism remained significant after Bonferroni correction and showed a significant association with endometrial cancer in both Asian- and European-ancestry samples.
Conclusions:
These findings lend support to the hypothesis that genetic polymorphisms in genes involved in the inflammatory pathway may contribute to genetic susceptibility to endometrial cancer. Impact statement: This study adds to the growing evidence that inflammation plays an important role in endometrial carcinogenesis.
... This indicates that βig-H3 processing is not mediated by a simple up-regulation of ovarian cancer cell derived proteases but requires multiple levels of cross-talk between both ovarian cancer and peritoneal cells. A similar paracrine effect was previously reported for endometrial cancer epithelium–stroma cell co-cultures, where hepatic growth factor secreted by the stromal cells acted on the endometrial cancer cells by inducing the cleavage of MMPs pro-forms to mature active forms [94]. Our findings suggest, however, that cleavage of βig-H3 in the ovarian cancer and peritoneal cell co-culture is not MMP mediated as the broad spectrum MMP inhibitor, GM6001, failed to inhibit βig-H3 processing. ...
Transforming growth factor-beta-induced protein (TGFBI, also known as βig-H3 and keratoepithelin) is an extracellular matrix protein that plays a role in a wide range of physiological and pathological conditions including diabetes, corneal dystrophy and tumorigenesis. Many reports indicate that βig-H3 functions as a tumor suppressor. Loss of βig-H3 expression has been described in several cancers including ovarian cancer and promoter hypermethylation has been identified as an important mechanism for the silencing of the TGFBI gene. Our recent findings that βig-H3 is down-regulated in ovarian cancer and that high concentrations of βig-H3 can induce ovarian cancer cell death support a tumor suppressor role. However, there is also convincing data in the literature reporting a tumor-promoting role for βig-H3. We have shown βig-H3 to be abundantly expressed by peritoneal cells and increase the metastatic potential of ovarian cancer cells by promoting cell motility, invasion, and adhesion to peritoneal cells. Our findings suggest that βig-H3 has dual functions and can act both as a tumor suppressor or tumor promoter depending on the tumor microenvironment. This article reviews the current understanding of βig-H3 function in cancer cells with particular focus on ovarian cancer.
... In endometrial cancer, it has been observed that HGF is secreted by endometrial stromal cells and that endometrial cancer cells express its receptor c-Met (14). The interaction between HGF and c-Met is involved in the effects of HGF on tumor invasion (14)(15)(16), angiogenesis (9) and anti-apoptosis (17). ...
Hepatocyte growth factor (HGF) is up-regulated in tissue repair and has been implicated in playing a role in this process through its anti-apoptotic and proliferative activities. Cyclooxygenase-2 (COX-2) is an inducible enzyme in the biosynthetic pathway of prostaglandins, and its activation has been shown to play an important role in cell growth. We previously reported that HGF significantly inhibited anoikis, possibly through the up-regulation of COX-2 expression in the endometrial RL95-2 cancer cell line. Here, we report that i) treatment of RL95-2 cells with HGF resulted in phosphorylation of the HGF receptor c-Met, activation of Akt and IκB, translocation of NF-κB into the nucleus, and up-regulation of COX-2 mRNA; ii) the IκB-α phosphorylation inhibitor BAY11-7082 and the selective COX-2 inhibitor CAY10452 blocked HGF-mediated anoikis resistance in RL95-2 cells; and iii) HGF induced migration and invasion in RL95-2 cells, while the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and CAY10452 blocked these effects of HGF stimulation. Our data suggest that HGF possesses chemotactic ability, has anti-apoptosis action, and induces cellular infiltration via the PI3K/Akt pathway; it also triggers NF-κB activation and up-regulates COX-2 gene expression in endometrial cancer cells.
... Several studies have suggested that increased expression of MMP-2 and MMP-9 may serve as biomarkers for the clinical behavior of endometrial cancers [58,59]. Moreover, MMP-2 and MMP-9 mRNA have been shown to be expressed by endometrial stromal cells but not expressed by endometrial cancer cell lines [60,61]. A study also showed that endometrial cancer cells demonstrate positive MMP-2 and MMP-9 staining via immunohistochemistry and in situ hybridization, with the intensity of staining correlated with histologic grade, disease progression and poor survival [62]. ...
Lysophosphatidic acid (LPA) has potent growth-regulatory effect in many cell types and has been linked to the in vivo tumor growth and metastasis in several malignancies. The goal of this study was to assess the regulation of (EC) microenvironment by LPA through the examination of its effect on cell proliferation, migration, invasion, uPA activity, and matrix metalloproteinase (MMP) secretion/activation.
All experiments were performed in vitro using an EC cell line, HEC-1A. Cell proliferation was determined using the Promega MTS proliferation assay following 48 h of exposures to different concentrations of LPA (0.1, 1.0 and 10.0 microM). Cell invasion was assessed using a modified Boyden chamber assay with collagen I coated on the membrane. HEC-1A motility was examined by Boyden chamber migration assay as well as the scratch wound closure assay on type I collagen. MMP secretion/activation in HEC-1A conditioned medium was detected by gelatin zymography. MMP-7 mRNA expression was determined using real-time PCR. uPA activity was measured using a coupled colorimetric assay.
LPA, at the concentrations of 0.1 and 1.0 microM, significantly induced the proliferation of HEC-1A cells (p<0.01). At 10 microM, LPA- induced HEC-1A proliferation to a less extent and showed no significant effect on HEC-1A invasion and migration (p>0.05). Gelatin zymogram showed that HEC-1A cells secreted high levels of MMP-7, while MMP-2 and MMP-9 are barely detectable. In addition, LPA significantly enhanced uPA activity in HEC-1A conditioned medium in a concentration-dependent manner.
LPA is a potent modulator of cellular proliferation and invasion for EC cells. It also has the capacity to stimulate the secretion/activity of uPA and MMP-7. Those results suggest that LPA is a bioactive modulator of EC microenvironment and may have a distinct regulation mechanism as observed in epithelial ovarian cancer.
... Lysosomale CPs (Cathepsin B und L) hingegen können pro-uPA aktivieren (Schmitt et al., 1997) und MMP-14; Kermorgant et al., 2001;Monvoisin et al., 2002;Bennett, et al., 2000;Fujiuchi et al., 2003;Harvey et al., 2000) und von Faktoren des Plasminogen-Aktivator-Systems (uPA, uPAR und PAI-1;Tacchini et al., 2004;Tacchini et al., 2003;Hall et al., 2004;Fujiuchi et al., 2003). Durch die Tatsache, dass dieser Signaltransduktionsweg sowohl zu einem Anstieg der MMP-Expression als auch Expression von Bestandteilen des PA-Systems führt, lässt sich die durch HGF verstärkte Aktivierung von proMMP-2 und -9 erklären (Park et al., 2003). Plasmin kann wie beschrieben proMMP-9 und MT1-MMP (MMP-14) aktivieren. ...
In the present study the concept of prophylactic overexpression of natural inhibitors of tumor-associated proteases (matrix metalloproteinases, plasminogen activators, cysteine proteases) by the host tissue to prevent experimental metastasis of a human fibrosarcoma cell line was investigated. Therefore, adenoviral vectors encoding for natural protease inhibitors (tissue inhibitor of metalloproteinases-1 (TIMP-1), plasminogen activator inhibitor-2 (PAI-2), and cystatin C, respectively) were constructed. Overexpression of these inhibitors significantly reduced the number of lung-metastases as compared to the control (by 93, 52, and 92%, respectively). A significant additive anti-metastatic effect was achieved by simultaneous inhibition of two proteolytic systems by TIMP-1 and PAI-2 (99% reduction). These results support the concept of prophylactic overexpression of natural protease inhibitors to prevent metastasis and demonstrate optimization of this concept by simultaneous inhibition of two or more tumor associated proteolytic systems.
... Several growth factors have been found to participate in MMP regulation in gynaecological cancers. Endothelin-1 (ET-1) (Rosanò et al. 2002) and HGF (Park et al. 2003) have been shown to increase both the secretion and activity of MMP-2 and MMP-9 in ovarian and endometrial cancer cell cultures, respectively. TGF-β/β1 has been found to increase MMP-2 expression in ovarian cancer cell lines (Lin et al. 2000) and MMP-2 and MMP-9 mRNA production in cervical cancer cell lines (Agarwal et al. 1994, Nuovo 1997). ...
... A PCR profile with initial denaturation for 2 min at 948C, followed by 45 s at 948C, 45 s at 508C, 45 s at 728C, 36 cycles (COX- 2), and a final extension step for 10 min at 728C was performed using a Genius thermal cycler (Techne, Inc., Cambridge, UK). The primer sequences and conditions for RT-PCR of MMP-2, MMP-9, TIMP-2, and MT1-MMP were according to Park et al.: Each PCR cycle consisted of 1 cycle for 3 min at 948C; 35 cycles for 30 s at 948C, 30 s at 578C, and 1 min at 728C; and 1 cycle for 5 min at 728C [20]. Amplification products were separated on a 1.5% agarose gel and stained with ethidium bromide. ...
Cyclooxygenase (COX)-2 is expressed in macrophages of arteriosclerotic lesions and promotes inflammation. We investigated whether COX-2 is already expressed in peripheral blood mononuclear cells (PBMCs) of subjects possessing risk-related factors, such as in smokers and hyperlipidemics. PBMCs were isolated from the venous blood of normolipidemic nonsmokers (NL-NSM; n = 15), normolipidemic smokers (NL-SM; n = 12), hyperlipidemic nonsmokers (HL-NSM; n = 10), and hyperlipidemic smokers (HL-SM; n = 10). RNA from PBMCs was used for RT-PCR. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA, those of glutamate and cystine by HPLC. The results show that COX-2 expression in PBMCs was significantly increased in the groups with cardiovascular risk factors (NL-SM, HL-SM, HL-NSM) compared with NL-NSM. COX-2 expression in PBMCs was positively correlated with concentrations of total serum cholesterol, oxLDL, glutamate, or cystine. We suggest that the elevated COX-2 expression indicates a priming of PBMCs as a response to a systemic pro-oxidative and proinflammatory shift in subjects with cardiovascular risk factors, which might also contribute to growth and instability of arteriosclerotic lesions.
... Expression of MMP-9 is regulated at transcription level and can be induced by classic mediators such as TNF-α (Yang et al., 2004), TGF-β (Rundhaug et al., 1997), EGF (Menashi et al., 2003) or HGF (Park et al., 2003). In our experiments, induction of MMP-9 did not require cell-cell contacts or viability of CA1a cells, indicating that it depends on one or more soluble factors already present in naive CA1a cells. ...
We used 2D-cocultures employing fibroblasts of different genetic backgrounds and MCF10A-derived human breast epithelial cells of increasingly malignant potential to investigate tumor-stroma interactions in breast cancer and to identify possible signaling pathways involved. Tumor cells induced expression of matrix-metalloproteinase 9 (MMP-9) in fibroblasts in a pattern dependent on the degree of their malignancy. In-situ zymography localized the main gelatinolytic activity around stromal cells in cocultures and xenografted tumors. Use of Smad3 knockout fibroblasts, small molecule inhibitors, and neutralizing antibodies showed that MMP-9 expression was induced by tumor cell-derived TNF-alpha and TGF-beta, dependent on Smad-, Ras-, and PI3-kinase-signaling, and likewise modulated by subsequent HGF- and EGF-signaling. Together, our results indicate that MMP-9 levels in tumor fibroblasts are regulated by a complex tumor-stroma cross-talk, involving multiple ligands and cellular signaling pathways.
... 39 Recent stud-ies also revealed that HGF promoted cancer invasion by activating MMP. 40,41 These studies support our findings, although the experiments were conducted under normoxic conditions. ...
The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1alpha expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1alpha expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1alpha expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.
... However, it is not known whether MMPs are exclusively involved in the proteolytic breakdown of the tissue for metastatic spread of the carcinoma. Previous studies have shown that MMPs were regulated by cytokines (23,24), transforming growth factor (25,26), hepatocyte growth factor (27) and ‚-catenin (25,28) in normal endometrium and endometrial carcinoma. On the other hand, the mRNA expression of MMP-7 and MMP-11 is reportedly regulated by ovarian steroid hormones (9,12,15,(29)(30)(31) alternating along with the menstrual cycle (32,33), although the expression of these proteins has not been confirmed. ...
Matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) are key factors in the degradation of extracellular matrix and basement membranes. This study aimed to examine the expressions of MMP-7 and -11 and TIMP-1 in normal, hyperplastic and neoplastic endometrium and their correlation to clinicopathologic factors.
Tissue samples of 40 normal endometria, 20 endometrial hyperplasias and 120 endometrial endometrioid adenocarcinomas were used for the study. Immunohistochemical staining for MMP-7 and -11 and TIMP-1 protein was performed on formalin-fixed and paraffin-embedded tissue samples. These expressions were represented as incidence of expression.
MMP-7 was highly expressed in the glands of the basal and functional layers during the proliferative and menstrual phases. MMP-11 expression in the gland of the basal layer and the stroma of the functional layer fluctuated during the menstrual cycle. TIMP-1 was highly expressed in the late secretory and menstrual phases. MMP-7 was expressed at significantly higher levels in endometrial hyperplasia than normal endometrium, whereas MMP-11 was expressed at lower levels. In endometrial adenocarcinoma, MMP-7, MMP-11 and TIMP-1 were expressed at the same levels as in hyperplasia. MMP-7 expression in endometrial carcinoma was correlated with myometrial invasion and estrogen receptor expression. The expression of MMP-7 in the adjacent stroma was associated with a poor prognosis.
MMP-7, MMP-11 and TIMP-1 expression may be regulated by the menstrual cycle, and related to the degradation and remodeling of the normal endometrium. MMP-7 expression might be a prognostic factor in endometrial carcinoma.
... Stromal-cell-rich fractions were purified, as described previously. 18,19 Primary stromal cells and HEC-1A cells were maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 media supplemented with 10% heat-inactivated FBS at 37°C and 5% CO 2 until use. ...
A novel cancer-cell-specific gene delivery vector with high transfection efficiency was designed and tested with an in vitro coculture consisting of the human endometrial adenocarcinoma cell line, HEC-1A cells, and normal endometrial stromal cells. For the cancer-cell targeting, polyethylenimine (PEI), a cationic polymer that can be easily combined with anionic DNA to form a particulate complex, polyplex, being capable of transferring a gene into a variety of cells, was covalently conjugated with antibodies against matrix metalloproteinase 2 (MMP-2), a typical surface-marker protein on cancer cells known for its close correlation with angiogenesis and invasion in many types of cancer, using the heterofunctional cross-linker, n-succinimidyl 3-(2-pyridyldithio)-propionamide. Biophysical properties and transfection efficiencies of anti-MMP-2-conjugated PEI were analyzed by means of dynamic light scattering, laser Doppler anemometry, and flow cytometry. Our results reveal that (1) the PEI-anti-MMP-2 antibody conjugate maintains physical parameters, including sizes and surface charges, which appear to be favorable for gene transfer and (2) when the pEGFP-N3 plasmid complexes of the PEI-anti-MMP-2 antibody conjugate are applied to the coculture consisting of HEC-1A cells and human stromal cells, a high level of green fluorescent protein expression occurs in HEC-1A cells over stromal cells, suggesting a specific gene transfer targeting cancer cells. Therefore, targeting invading cancer cells with the PEI-anti-MMP-2 antibody conjugate could be promising in endometrial cancer treatment, and this gene delivery system deserves further optimization in the context of targeted therapeutic gene delivery.
Background
Matrix metalloproteinases (MMPs) play a fundamental role in tissue degradation or remodeling and involved in all stages of tumor progression. However, the role of the MMPs polymorphisms with endometrial carcinoma has not been fully examined in Egyptian patients. Therefore, we planned this study to evaluate associations of the MMP-1 (− 1607 1G/2G) and MMP-3 (− 1171 5A/6A) polymorphisms and their immune reactive protein combinations with endometrial carcinoma susceptibility and prognosis in Egyptian patients.
Methods
Paraffin-embedded tissue samples from 40 women with endometrial carcinoma and 30 controls were immunohistochemically stained for MMP-1and MMP-3 expression. Tissue MMPs levels were analyzed by ELISA technique, and tissue samples were genotyped for MMP-1 and MMp-3 gene polymorphisms by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP).
Results
we recorded a significant increase of MMP-1 levels in endometrial carcinoma patients more than controls, to be more increased with advanced stages and grades of the carcinoma. Whereas, the MMP-3 tissue levels showed non-significant changes among patients and controls and even among different stages and grades of cancer.
Patients with endometrial carcinoma exhibited a higher distribution of MMP-11G/2G or 2G/2G genotypes compared with controls. Tumors containing the 2G allele expressed MMP-1 protein more frequently than those of 1G/1G genotype. The overall genotype and alleles distribution of the MMP-3 polymorphism in patients was not different from that of controls. The haplotype 2G-6A was associated with a significantly increased risk of endometrial carcinoma as compared with the 1G-5A haplotype.
Conclusion
The MMP-1 (− 1G/2G) SNP and MMP 2G/6A haplotype may modify susceptibility and are associated with a higher risk of endometrial carcinoma. Otherwise, the MMP-3 (5A/6A) promoter SNP is unlikely to be associated with endometrial carcinoma in the Egyptian population.
Endometrial cancer (EC) is the most common gynecologic malignancy in the western world with more than 280,000 cases per year worldwide. Prognosis for EC at early stages, when primary surgical resection is the most common initial treatment, is excellent. Five-year survival rate is around 70 %.
Several molecular alterations have been described in the different types of EC. They occur in genes involved in important signaling pathways. In this chapter, we will review the most relevant altered pathways in EC, including PI3K/AKT/mTOR, RAS–RAF–MEK–ERK, Tyrosine kinase, WNT/β-Catenin, cell cycle, and TGF-β signaling pathways. At the end of the chapter, the most significant clinical trials will be briefly discussed.
This information is important to identify specific targets for therapy.
Endometrial cancer (EC) is one of the most common gynaecological malignancy. Unfortunatelly, molecular pathogenesis of this neoplasm remains poorly understood. More than 90% od EC cases are sporadic and they can be divided into two main subgroups. The first group consists of oestrogen-related tumours that occur in pre and post-menopausal women and are frequently proceded by endometrial hyperplasia. These tumours are usually associated with abnormalities of DNA-mismatch repair genes, k-ras, PTEN and beta-catenin. The second group consists of tumours that occurs mainly in post-menopausal women and which are usually not related to oestrogen. These tumours are characterized by abnormalities of p53 and HER2/neu. The aim of our study was to profile expression of cellular receptor genes to candidate those typical for tumor grade or clinical stage by the mean of macro Array technique. Based on Kruskal-Wilis test we pointed out nine genes encoding cellular receptor proteins showing statistically significant over or under-expression (4 and 5 genes respectively) depending tumor grade. The analysis of statistic dependences between gene expression profiles and clinical stage of tumor pointed out 15 genes encoding cellular receptor proteins the expressions of which were altered showing statistical significance for different clinical stages. It is worthy to note that understanding of mechanisms underlying alternation of genes expression is a real key to establish new prognostic factors and tailored therapy for endometrial cancer.
Hepatocyte growth factor (HGF) regulates cell growth, motility, and morphogenesis of various types of cells via its receptor c-Met. In the growth of endometrium, HGF/c-Met signaling promotes the proliferation of both endometrial epithelial cells and stromal cells. In the onset and development of several cancers, HGF/c-Met signaling drives carcinogenesis and cancer invasion and metastasis. In endometrial carcinoma (EC), accounting for a majority of cancer-related morbidity and mortality among women, the increased expression of HGF and c-Met signaling is associated with a poor prognosis of EC patients. Thus, we will discuss the role of HGF and c-Met in the EC pathogenesis in this review.
The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.
An effort was undertaken to identify phenolic acids contained in the water sample remaining from steam distillation of the volatile fraction contained in winter savory (Satureja montana). Solid phase extraction (SPE) was employed to isolate the phenolics from water. Additionally, the accelerated solvent extraction (ASE) was used to isolate the phenolics from the dried winter savory material. The phenolics derived both from the water residue and those obtained with use of ASE were first derivatized and then analyzed by means of GC/MS. The non-derivatized phenolics originating from ASE were fingerprinted and semi-quantitatively assessed by means of HPLC/DAD. A comparison was made of the GC and HPLC results, and the conclusions were drawn as to the identified compounds, depending on the applied analytical approach. Usefulness of the complementary approach was shown, as each of these two techniques allowed detection of the different phenolic acids.
Hepatocellular carcinoma (HCC) is closely associated with chronic liver diseases, particularly cirrhosis. However, the genes involved in hepatocarcinogenesis in the context of developing cirrhosis remain unknown. This study aims to identify genes associated with early cirrhosis-associated hepatocarcinogenesis.
We examined genes differentially expressed between the livers of normal rats and rats fed a choline-deficient, L-amino acid-defined (CDAA) diet using suppression subtractive hybridization. We examined both the expression in the liver and HCC tissues of osteoactivin (OA), isolated in this screen, and its effect on invasiveness and metastasis.
OA mRNA was strongly expressed in the livers of rats fed the CDAA diet for 1-3 months. Moderate expression was sustained for 18 months. OA overexpression increased the invasiveness and metastasis of rat hepatoma cells in vitro and in vivo. In humans, OA expression was not detectable in normal liver tissues. While OA transcripts were detectable in cirrhotic nontumorous liver tissues surrounding HCCs, the majority of HCC tissue samples exhibited higher levels of OA expression than the surrounding normal tissue.
These results indicate that OA is a novel factor involved in the progression of HCC via stimulation of tumor invasiveness and metastatic potential.
In recent video applications such as MPEG or H.264/AVC, the bandwidth requirement for frame memory has become one of the most critical problems. Compressing pixel data before storing in off-chip frame memory is required to alleviate this problem. In this paper, we propose a lossless frame memory recompression scheme including 1) a lossless pixel compression algorithm, 2) an efficient address table organization method for random accessibility, and 3) frame memory placement scheme for compressed data to reduce the effective access time of SDRAM by suppressing row switching. Experimental results show that the proposed method reduces the frame data to 48% compared to that of the uncompressed one with H.264/AVC high profile encoder system, where 6.1 kB of SRAM is required for the address table of full HD video.
Ovarian cancer metastasis is characterized by the shedding of malignant cells from the surface of the ovary and their implantation onto the peritoneal surface, which lines the abdominal cavity. As the factors promoting this process are poorly understood, we investigated the ovarian cancer-peritoneal interaction by means of in vitro coculture experiments with ovarian cancer (OVCAR-5 and SKOV-3) and peritoneal (LP-9) cells. One of the proteins differentially expressed in the coculture secretome was identified by MALDI-TOF/TOF mass spectrometry as the extracellular matrix protein transforming growth factor-beta-induced protein (TGFBIp, also known as βig-H3). Immunohistochemistry showed high TGFBIp levels in normal surface ovarian epithelial and peritoneal cells, whereas TGFBIp levels in primary serous ovarian carcinomas and matching metastatic implants was very low. In functional in vitro experiments, treatment with recombinant TGFBIp significantly increased the motility and invasiveness of OVCAR-5 and SKOV-3 cells and significantly increased ovarian cancer cell (OVCAR-5, OVCAR-3 and SKOV-3) adhesion to LP-9 cells. TGFBIp was found to be processed at both the N- and C-terminus in the secretome of the ovarian cancer-peritoneal cell coculture. Plasmin inhibitors blocked TGFBIp processing and significantly reduced OVCAR-5 cell adhesion to peritoneal cells. We conclude that TGFBIp expressed by peritoneal cells increases the metastatic potential of ovarian cancer cells. TGFBIp is therefore a potential novel therapeutic target against ovarian cancer.
MMPs are a group of zinc-dependent endopeptidases implicated in the degradation of extracellular matrix components. Angiogenesis, wound healing and tissue remodeling are the main processes in which MMPs have been implicated. MMPs also seem to play a role in the pathophysiological processes during tumorigenesis and are thought to have predictive value for clinical outcome. Alterations in the regulation of MMP expression are considered to be of significant importance in the development and progression of gynecological malignancies.
This study provides a literature review on the involvement of MMPs in the development and progression of gynecological malignancies, their clinical significance and the potential use of natural and/or synthetic MMP Inhibitors (MMPIs) as a more targeted therapeutic approach to these neoplasms.
There is mounting evidence that MMPs play an important role in development and progression of gynecological malignancies. Current results provide the basis for further investigation of their role in malignancies, the therapeutic potential of their inhibition and the side-effects that can be expected from the modulation of matrix remodeling during therapeutic intervention. Focused research on the combination of MMPIs with cytotoxic and/or antiangiogenic compounds might provide the key to more successful therapeutic approaches towards gynecological and other malignancies.
Up-regulated expression of syndecan-1, a member of the transmembranous proteoglycans that serves as a co-receptor for a wide pool of extracellular ligands, has been ascribed to the promotion of growth of various cancers including breast, ovarian, and endometrial cancers. Here, we have extended these observations to gain insight into correlation between the expression level of syndecan-1 and its tumor-promoting characteristics, particularly, cancer invasion, in endometrial cancer.
Human syndecan-1 was stably transfected into three human endometrial cancer cell lines, and its effects were examined with respect to cell survival/proliferation and invasion. In addition, the activation of underlying signaling components, including integrins, focal adhesion kinase (FAK), and nuclear factor kappaB (NF-kappaB) was examined. The activity of NF-kappaB as a transcription factor for matrix metalloproteinase (MMP)-9 was assessed.
The innate expression level of syndecan-1 was moderate to high in all endometrial cancer cell lines. Overexpression of syndecan-1 promoted tumor cell proliferation concomitant with the activation of NF-kappaB. Furthermore, overexpression of syndecan-1 markedly enhanced the cancer invasion accompanied by enhanced expression of integrin alphav/beta5 and enhanced phosphorylation of FAK. The transcriptional activation of MMP-9 by NF-kappaB was up-regulated in syndecan-1 overexpression.
These findings provide evidence that supports that syndecan-1 may have a critical role in carcinogenic progression, particularly, contributing to the development of proliferative and invasive phenotype through NF-kappaB-mediated MMP-9 gene expression in endometrial cancer.
Junctional adhesion molecule A (JAM-A) is involved in cell-cell contact and tight junction formation. Loss of cell adhesion molecules may be associated with high histologic grade and invasiveness of endometrial carcinoma. We attempted to determine JAM-A expression in human endometrial carcinoma and its correlations with pathologic features, stage, and survival. Junctional adhesion molecule A expression in human endometrial carcinoma was evaluated by immunohistochemistry. In addition, we cultured human well and poorly differentiated endometrial adenocarcinoma cell lines, Ishikawa cells, and KLE in 3-dimensional basement membrane preparation, and JAM-A expression in these cells was assessed by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Junctional adhesion molecule A immunostaining intensity was negatively correlated with histologic grade (tau = -0.420, P < 0.0001), myometrial invasion (tau = -0.306, P < 0.01), and stage (tau = -0.383, P < 0.0001). Low JAM-A immunostaining intensity was associated with positive vascular space involvement (P < 0.01). Moreover, low immunostain intensity was significantly (P < 0.0001) related to low overall survival rate and progression-free survival rate. Additionally, in our 3-dimensional epithelial cell culture, JAM-A expression in poorly differentiated adenocarcinoma was significantly lower than that in well-differentiated adenocarcinoma (P < 0.001). Junctional adhesion molecule A expression seems to be reduced in high-grade or advanced endometrial carcinoma and may be a prognostic factor.
The adipocytokines are biologically active polypeptides that are produced either exclusively or substantially by the adipocytes, and act by endocrine, paracrine, and autocrine mechanisms. Most have been associated with obesity, hyperinsulinaemia, type 2 diabetes, and chronic vascular disease; in addition, six adipocytokines--vascular endothelial growth factor, hepatocyte growth factor, leptin, tumour necrosis factor-alpha, heparin-binding epidermal growth factor-like growth factor, and interleukin-6--promote angiogenesis while one, adiponectin, is inhibitory. Obesity and insulin resistance have both been identified as risk factors for breast cancer and are associated with late-stage disease and poor prognosis. Angiogenesis is essential for breast cancer development and progression, and so it is plausible that obesity-related increases in adipocytokine production and a reduction in adiponectin may adversely affect breast cancer outcome by their angiogenesis-related activities. There is also experimental evidence that some adipocytokines can act directly on breast cancer cells to stimulate their proliferation and invasive capacity. Thus, adipocytokines may provide a biological mechanism by which obesity and insulin resistance are causally associated with breast cancer risk and poor prognosis. Both experimental and clinical studies are needed to develop this concept, and particularly in oestrogen-independent breast cancers where preventive and therapeutic options are limited.
Hepatocyte growth factor (HGF) stimulates the migration of myogenic cells during the development of skeletal muscles. The inactivation of HGF genes or that of its receptor, c-met, in mice causes hypoplasia of skeletal muscle organs, such as the tongue. Basic fibroblast growth factor (FGF-2) also induces migration of skeletal myoblasts. A comparison of the functions of HGF and FGF-2 in myogenesis revealed the crucial effect of HGF in the development of skeletal muscles. Unlike FGF-2, HGF induced migration of myoblasts from the developing mouse tongue. The differences between the activities of HGF and FGF-2 were determined by comparing their effects on the expression of matrix metalloproteinase-9 (MMP-9) in myoblasts, C2C12 cells, cultured in collagen-coated dishes. The results showed that HGF, but not FGF-2, stimulated MMP-9 expression, and that the stimulation was mediated through the activation of phosphoinositide 3-kinase (PI3K) which was not associated with FGF-2 signal transduction. Nevertheless, both growth factors exerted almost the same effect on the reduction of myogenin expression in, and on the proliferation of, C2C12 cells, suggesting that HGF, rather than FGF-2, plays a crucial role in the generation of skeletal muscles, including the tongue. Moreover, the specific role of HGF through the PI3K signal pathway is the induction of MMP-9 expression in, and the migration of, myoblasts.
Cyclooxygenase-2 (COX-2) has been implicated in the promotion of carcinogenesis. Although the role of COX-2 in endometrial cancer remains unclear, recent experiments suggest that COX-2 antagonizes cell apoptosis, increases the invasiveness of malignant cells, and promotes angiogenesis. Hepatocyte growth factor (HGF) is a mesenchymal-derived cytokine and the interaction between HGF and its tyrosine kinase receptor, c-Met proto-oncogene, is associated with tumor progression and metastasis. To investigate the molecular mechanism of HGF-induced anoikis resistance, we analyzed the signal transduction and COX-2 expression in endometrial cancer cells. Here, we show i) the expression of COX-2 protein significantly increased in a dose-dependent manner after HGF stimulation in endometrial cancer cell lines (HEC-IB and RL95-2), reaching 200-270% stimulation at the highest doses of HGF tested (40 ng/ml); ii) flow cytometry and TUNEL analyses revealed that HGF significantly inhibited anoikis of RL95-2 cells; iii) phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), but not mitogen-activated protein kinase/ERK kinase (MEK) inhibitor (PD98059), specifically blocked HGF-mediated anoikis resistance in RL95-2 cells; and iv) COX-2 inhibitor, Meloxicam, abrogated HGF-mediated anoikis resistance. Our data suggest that HGF induces anoikis resistance in endometrial cancer cells possibly through PI3K/Akt pathway-dependent up-regulation of COX-2 expression.
To determine the role of peritoneal mesothelial cells (PMCs) in the process of endometrial invasion into the peritoneum and to evaluate gene expression after endometrial-PMC co-culture.
In vitro study.
University laboratory.
Reproductive-age women without endometriosis.
None.
The rate of endometrial invasion through modeled peritoneum in the presence and absence of PMCs was evaluated. The influence of endometrial-PMC attachment on the expression of target genes, implicated in the pathogenesis of endometriosis, was examined by using reverse transcription polymerase chain reaction.
Endometrial stromal cell (ESC) invasion through invasion chambers coated with Matrigel (MTGL) and with growth factor-reduced Matrigel (GFR-MTGL) was increased 10-fold when a PMC monolayer was present. Endometrial epithelioid cell (EM42) invasion increased greater than threefold through the MTGL and GFR-MTGL-coated membranes when a PMC monolayer was present. Endometrial stromal cell, EM42, and PMC transcription of extracellular signal-related kinase, colony stimulating factor-1, c-fms, and c-Met was increased after endometrial-PMC attachment. Similar changes were not seen when endometrial cells were exposed to PMC-conditioned media and when PMCs were exposed to endometrial cell conditioned media.
Peritoneal mesothelial cells increased invasion of ESCs and EM42s through modeled peritoneum. Endometrial-PMC co-culture led to alterations in gene transcription by endometrial cells and PMCs. This study suggests that PMCs contribute to the process of endometrial invasion into the peritoneum.
H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.
The specificity of human skin collagenase and of an enzyme from an invasive tumor were studied by using types I, II, III, IV, and V (AB) collagen as substrates. Human skin collagenase degraded types I, II, and III collagen, producing the characteristic 3/4 and 1/4 cleavage products, but failed to degrade type IV or V collagen. Collagenase prepared from the invasive tumors showed maximal activity after trypsin treatment. The tumor enzyme degraded type IV (basement membrane) collagen, producing fragments consistent with a single cleavage site but did not attack types I, II, III, and V collagen. Because type IV collagen prepared by pepsinization of placenta was also digested, it is likely that cleavage of type IV collagen by the tumor collagenase occurs within a largely helical domain. A type IV collagenase could play a significant role in tumor metastases and in normal tissues where basement membrane turnover takes place.
We have examined the expression of 2 type IV collagen degrading enzymes (Mr 72,000 and 92,000 type IV collagenases) in human skin cancer by in situ hybridization. In all cases of infiltrating carcinomas of squamous cell (9 of 9) and basal cell (5 of 5) types, messenger RNA for the Mr 72,000 type IV collagenase was present in numerous fibroblasts. These were especially abundant in the stroma adjacent to the invasive tumor nodules. Malignant cells were negative for mRNA for the Mr 72,000 enzyme in all cases as were all other epithelial as well as endothelial cells. mRNA for the Mr 92,000 type IV collagenase was present in all 9 squamous cell and in 3 of the 5 basal cell carcinomas. In all these cases, a subpopulation of tissue macrophages was found to be positive, while malignant cells showed a signal for Mr 92,000 type IV collagenase in 6 of the squamous cell carcinomas but in none of the basal cell carcinomas. In all cases, the signal for this mRNA was confined to cells located at the tumoral/stromal interface or in the close vicinity of tumor nodules. No mRNA for any of the 2 collagenases was detected in 3 biopsies of normal skin. In vitro studies have indicated that collagenases are involved in the degradation of the extracellular matrix during cancer invasion. The present findings are consistent with such a role of the Mr 72,000 and 92,000 type IV collagenases in squamous and basal cell carcinomas in situ. The findings also demonstrate that degradative enzymes are not necessarily produced by the malignant cells themselves but may be generated by induction or recruitment of nonmalignant stromal cells.
The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.
We isolated overlapping cDNA clones corresponding to the major MET protooncogene transcript. The cDNA nucleotide sequence contained an open reading frame of 1408 amino acids with features characteristic of the tyrosine kinase family of growth factor receptors. These features include a putative 24-amino acid signal peptide and a candidate, hybrophobic, membrane-spanning segment of 23 amino acids, which defines an extracellular domain of 926 amino acids that could serve as a ligand-binding domain. A putative intracellular domain 435 amino acids long shows high homology with the SRC family of tyrosine kinases and within the kinase domain is most homologous with the human insulin receptor (44%) and v-abl (41%). Despite these similarities, however, we found no apparent sequence homology to other growth factor receptors in the putative ligand-binding domain. We conclude from these results that the MET protooncogene is a cell-surface receptor for an as-yet-unknown ligand.
H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.
Fibroblasts or their conditioned medium stimulated invasion by squamous cell carcinoma cells. The fibroblast-derived activity responsible for increased invasion is the hepatocyte growth factor/scatter factor (HGF/SF), a ligand for the c-Met receptor. HGF/SF stimulated migration of the cells on various extracellular matrix substrates but did not alter their adhesion efficiency nor integrin expression. HGF/SF stimulated motility in a two step process: initially cells spread rapidly and formed focal adhesions, and then they disassembled these condensations, which was followed by increased cell locomotion. The focal adhesions contained vinculin, p125FAK, beta 1 integrin, and phosphotyrosine. Within minutes after exposure of cells to HGF/SF, proteins of 125 and 145 kDa showed elevated tyrosine phosphorylation and were identified as p125FAK and c-Met, respectively. Gradual loss of tyrosine phosphorylation coincided with disruption of focal adhesions and conversion to a motile phenotype. HGF/SF-mediated tyrosine phosphorylation of p125FAK was inhibited by the tyrosine kinase inhibitor, herbimycin A, which also blocked spreading and the migratory response. These results indicate that fibroblast-derived HGF/SF triggers migration through the initial recruiting of integrins, cytoskeletal proteins, and p125FAK into focal adhesions that is dependent on tyrosine kinase activity.
Matrix metalloproteases are secreted by mammalian cells as zymogens and, upon activation, initiate tissue remodeling by proteolytic degradation of collagens and proteoglycans. Activation of the secreted proenzymes and interaction with their specific inhibitors determine the net enzymatic activity in the extracellular space. We have previously demonstrated that 72T4Cl can be activated by a plasma membrane-dependent mechanism specific for this enzyme. Here, we report purification of the membrane activator of 72T4Cl, which is a new metalloprotease identical to a recently cloned membrane-type matrix metalloprotease (MT-MMP). We demonstrate that activated MT-MMP acts as a cell surface tissue inhibitor of metalloprotease 2 (TIMP-2) receptor with Kd = 2.54 x 10(-9) M. The activator.TIMP-2 complex in turn acts as a receptor for 72T4Cl (Kd = 0.56 x 10(-9) M, binding to the carboxyl-end domain of the enzyme. Activation of 72T4Cl on the cell membrane provides a basic mechanism for spatially regulated extracellular proteolysis and presents a new target for prognosis and treatment of metastatic disease. The activation, purified as a tri-molecular complex of MT-MMP.TIMP2.carboxyl-end domain of 72T4Cl, is itself an activated form of MT-MMP, posing the following question: what is the mechanism of the activator's activation?
The met proto-oncogene is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is a multifunctional cytokine that stimulates mitogenesis, motility, invasion, and tubulogenesis of a spectrum of epithelial and endothelial cells in culture. Using a chimeric receptor (CSF-MET), containing the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domain of the Met receptor, we have previously demonstrated that activation of the Met kinase domain is sufficient to mediate the motility, invasion and morphogenic signals of HGF/SF in Madin-Darby canine kidney epithelial cells (MDCK). In this study we have analyzed the role of tyrosine phosphorylation of the Met receptor in the transmission of these signals by site-directed mutagenesis of specific tyrosine residues. Mutation of two tyrosine residues (tyrosine 1234 and tyrosine 1235), involved in activation of the catalytic activity of the kinase, abrogates the biological activity of the chimera. In addition, we have identified a single noncatalytic tyrosine residue (tyrosine 1356) in the carboxyl terminus of the Met receptor, that is essential for the biological activity of the chimeric receptor. Mutation of tyrosine 1356 to a nonphosphorylatable phenylalanine residue does not affect the exogenous kinase activity of the receptor toward enolase, but it impairs the ability of the mutant protein to associate with the adaptor protein Grb2, and MDCK cells expressing this mutant fail to scatter, invade, and form branching tubules in response to CSF-1. These results support a crucial role for tyrosine 1356 in activation of signaling pathways involved in the biological activity of the Met receptor in response to HGF/SF.
Using zymography and computer assisted image analysis, we have measured the levels of type IV collagenases in biopsies from normal breast, and benign and malignant breast disease. The 92 kDa form was present in three of 11 cases of normal/benign disease, three of nine grade I tumours, four of 12 grade II tumours, but 11 of 11 grade III tumours. Mean levels were higher in grade III tumours (P < 0.0001). When the levels of 72 kDa collagenase and its active 62 kDa form were considered together, there was no difference between the benign and malignant cases (P = 0.55), but the amount of active enzyme, considered as a proportion of the 62 + 72 kDa forms, was significantly higher in malignant disease (P = 0.003). There was also a trend towards a higher proportion of active enzyme with increasing tumour grade (P < 0.0001). In situ hybridisation and immunohistochemistry studies showed that that mRNA and protein for the 92 kDa enzyme was primarily found in the tumour stroma. mRNA for the 72 kDa enzyme was also found in stromal areas. This study demonstrates a clear relationship between production of Type IV collagenases and malignant breast disease. Inhibitors of these enzymes may be of value in preventing metastatic disease.
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Figure 1
Figure 2
Matrix metalloproteinases (MMPs), which degrade the components of the extracellular matrix, are key enzymes involved in the tissue remodeling of multicellular organisms. Since MMPs are secreted as inactive zymogens (pro-MMPs), they have to be activated to function. We identified a membrane-type MMP (MT-MMP) that activated proMMP-2 (pro-gelatinase A = 72 kDa type IV pro-collagenase) and described its expression on the invasive tumor cell surface. In this study we further examined the expression and role of MT-MMP in the activation of proMMP-2 during mouse embryogenesis. Northern blotting demonstrated that MT-MMP expression was increased together with that of MMP-2 and its inhibitor gene, TIMP-2, in embryos depending upon the number of days after gestation, and decreased with maturation after birth. In situ hybridization and immunohistochemistry localized MT-MMP mRNA and protein in the cells of ossifying tissues where both MMP-2 and TIMP-2 were expressed. Activated MMP-2 was detected by gelatin zymography in the lysates prepared from the micro dissected tissues that expressed the three genes. The activation rate of proMMP-2 was proportional to the expression of MMP-2 and MT-MMP. These results indicated that proMMP-2 activation through its activator, MT-MMP, is a physiological system used by organisms to initiate tissue remodeling on the cell surface.
The matrix metalloproteinases (MMPs) are perceived as essential for tumour invasion and metastases. The purpose of this study was to determine the expression and cellular localisation of the 92 kDa type IV collagenase (MMP-9) protein and mRNA in human colorectal cancer (CRC). In CRC and matched normal mucosa specimens from 26 CRC patients, Northern blot hybridisation and Western blot analyses provide convincing evidence that MMP-9 is expressed in greater quantities in CRC than in normal tissue. The MMP-9 tumour to normal mucosa fold-increase (T/N) was 9.7 +/- 7.1 (mean +/- s.d.) (P < 0.001) for RNA and 7.1 +/- 3.9 (P < 0.001) for protein. The sites of MMP-9 mRNA and protein synthesis were colocalised in tumour stroma by in situ hybridisation and immunohistochemistry in 26 CRC samples. Both MMP-9 mRNA and protein signals were strongest in the population of stromal cells concentrated at the tumour-stroma interface of an invading tumour. Furthermore, MMP-9-positive cells were identified as macrophages using an antimacrophage antibody (KP1) in serial sections from ten CRC samples. Given the persistent localisation of MMP-9-producing macrophages to the interphase between CRC and surrounding stroma, our observations suggest that MMP-9 production is controlled, in part, by tumour-stroma cell interactions. Further studies are needed to determine the in vivo regulation of MMP-9 production from infiltrating peritumour macrophages.
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Figure 1Figure 2Figure 3Figure 4Figure 5
Activated gelatinase A is reportedly associated with tumor spread. We identified novel matrix metalloproteinases that localize
on the cell surface and mediate the activation of progelatinase A. Thus, these progelatinase A activators were named membrane-type
matrix metalloproteinase-1 and −2 (MT-MMP-1 and −2, respectively). MT-MMP-1 is overexpressed in malignant tumor tissues, including
lung and stomach carcinomas that contain activated gelatinase A. This suggests that MT-MMP-1 is associated with the activation
of progelatinase A in these tumor tissues. The expression of MT-MMP-1 also induced binding of gelatinase A to the cell surface
by functioning as a receptor. The cell surface localization of proteinases has advantages over pericellular proteolysis. MT-MMP
-1 and its family may play a central role in the cell surface localization and activation of progelatinase A and via this mechanism, tumor cell use exogenous progelatinase A to mediate the proteolysis associated with invasion and metastasis.
It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an
intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on
the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding,
we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on
COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells
with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition
of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds
to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2,
the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation
using antibodies to MT1-MMP were employed to identify 125I-TIMP-2·MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin
A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.
Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP. In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2. The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner. In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations. TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA. A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available. Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F.
Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.
: Tumor progression is a complex, multistage process by which a normal cell undergoes genetic changes that result in phenotypic alterations and the acquisition of the ability to spread and colonize distant sites in the body. Although many factors regulate malignant tumor growth and spread, interactions between a tumor and its surrounding microenvironment result in the production of important protein products that are crucial to each step of tumor progression. The matrix metalloproteinases (MMPs) are a family of degradative enzymes with clear links to malignancy. These enzymes are associated with tumor cell invasion of the basement membrane and stroma, blood vessel penetration, and metastasis. They have more recently been implicated in primary and metastatic tumor growth and angiogenesis, and they may even have a role in tumor promotion. This review outlines our current understanding of the MMP family, including the association of particular MMPs with malignant phenotypes and the role of MMPs in sp...
Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase 3 (M(r) 60,000) to the fully active enzyme (Tyr(85) N terminus; M(r) 48,000). MT1-MMP activated procollagenase-3 via a M(r) 56,000 intermediate (Ile(36) N terminus) to 48,000 which was the result of the cleavage of the Glu(84)-Tyr(85) peptide bond. We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family. In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys(38)-GlU(39) and Arg(76)-Cys(77) peptide bonds in the propeptide domain, Autoproteolysis then resulted in the release of the rest of She propeptide domain generating Tyr(85) N-terminal active collagenase 3. However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity. Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a M, 56,000 intermediate form to the final M(r) 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-a, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation, We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin A-stimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.
Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin αvβ3. MMP-2 and αvβ3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of αvβ3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind αvβ3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.
We have studied in vitro the effect of a hydroxyapatite (HA) tricalcium phosphate material coated with hepatocyte growth factor (HA-HGF) on cell growth, collagen synthesis and secretion of metalloproteinases (MMPs) by human osteoblasts, Cell proliferation was stimulated when osteoblasts were incubated with untreated HA and was further increased after exposure to HA-HGF, The uptake of [H-3]-proline was increased after treatment with HA. When osteoblasts were exposed to HA-HGF, collagen synthesis was increased with respect to HA. The secretion of MMPs in control cells was undetectable, but in HA and HA-HGF cells MMP 2 and MMP 9 were clearly synthesised. Our results suggest that HA can promote osteoblast activity and that HGF can further increase its bioactivity.
Collagen fibres form the stable architecture of connective tissues and their breakdown is a key irreversible step in many pathological conditions1,2. The destruction of collagen is usually initiated by proteinases, the best known of which is the metalloproteinase collagenase (EC 3.4.24)3. Collagenase and related metalloproteinases are regulated at the level of their synthesis and secretion, through the action of specific stimuli such as hormones and cytokines, and also at the level of their extracellular activity through the action of a specific inhibitor, TIMP (tissue inhibitor of metalloproteinases)4–6, which irreversibly forms inactive complexes with metalloproteinases7. Although the mechanisms governing the production of TIMP are unknown, immunologically identical forms of this glycoprotein have been detected in a wide variety of human body fluids and cell and tissue culture media5,8. We therefore suggested5 that under physiological conditions this ubiquitous inhibitor predominates over active metalloproteinases and that tissue destruction may arise when any perturbation of this controlling excess arises. However, further progress towards testing this theory has been hindered by a lack of knowledge about the structure of TIMP and insufficient material for studying it in model systems. Here we describe the structure of TIMP predicted from its complementary DNA, its synthesis in Escherichia coli and transfected animal cells, and the finding that it is identical to a protein recently reported to have erythroid-potentiating activity (EPA)9.
We examined the in situ distribution of basement membrane collagen (Col IV), matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) by immunohisto-chemistry and their mRNA levels by Northern blot analysis in 14 cases of squamous cell carcinoma of the lung. Elevated mRNA levels of MMP-2 and Col IV were demonstrated in all the cases examined and were associated with in situ disruption of basement membranes around the tumor nests. In contrast, TIMP-1 mRNA levels were not altered. MMP-2, MMP-9 and TIMP-1 were localized in tumor cells, stromal fibroblasts and endothelial cells. There were no significant correlations between these parameters and clinical staging. The results suggest that the degrading enzymes of basement membrane collagen play an important role in the invasion and metastasis of human squamous cell carcinoma of the lung.
Thyroid cancer can degrade basement membranes and invade tissues. This depends on a cascade of matrix metalloproteinases involving membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, and MMP-9. We analyzed the expression and role of these MMPs and their specific inhibitors TIMP-2 and TIMP-3 in human highly purified thyroid epithelial, C 643, HTh 74, SW 1736, and 8505 C thyroid carcinoma and thyroid-derived fibroblast cell cultures. The effect of phorbol-myristate acetate (PMA), and of the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) on MMP and TIMP mRNA levels were monitored by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) including an internal homologous competitor fragment. The highest MT1-MMP mRNA levels were found in thyroid-derived fibroblasts. The MT1-MMP mRNA expression was increased up to 10-fold by PMA, while all other growth factors tested had only negligible effects. The thyroid carcinoma cells themselves did not seem to play a crucial role in the production of MT1-MMP in thyroid tumors. Higher MMP-2 mRNA levels were found in all cell types investigated. The highest MMP-2 mRNA levels were determined in thyroid-derived fibroblasts and HTh 74 cells. We found a lack of MMP-2 response to IL-1, TNF-alpha, and phorbol esters. In unstimulated cells, MMP-9 mRNA was found near the detection limit or at low levels. In nearly all cell types, treatment with PMA, IL-1, and TNF-alpha caused an increase of the MMP-9 mRNA levels. The results of basal and stimulated MMP-2 and MMP-9 mRNA expression were confirmed at the protein level by gelatin zymography. TIMP-2 and TIMP-3 mRNAs were expressed at high levels. In contrast to the basal TIMP-3 mRNA levels, which varied over a great range, there were no striking differences the cell types from analyzing TIMP-2 mRNA. There were no or only slight stimulatory effects on TIMP-2 and TIMP-3 mRNA expression by IL-1, TNF-alpha, and PMA. Taken together, most enzymes of the MT-MMP/MMP class of proteases facilitating invasion of thyroid tumor cells seem to have been produced by fibroblasts, not by the tumor cells themselves. However, some dedifferentiated thyroid tumor cell lines may be capable of secreting some of these enzymes, as in the case of HTh 74 cells.
Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.
A trihexacontapeptide corresponding to the sequence 8-70 of eglin c and its related peptides were synthesized by the conventional solution method and their inhibitory activity against human leukocyte elastase, cathepsin G and alpha-chymotrypsin was examined. Although synthetic eglin c (41-49) inhibited cathepsin G and alpha-chymotrypsin (Ki = 4.0 x 10(-5) M and 2.0 x 10(-5) M, respectively) but not leukocyte elastase, the synthetic trihexacontapeptide potently inhibited cathepsin G, alpha-chymotrypsin and leukocyte elastase (Ki = 1.8 x 10(-9) M, 1.4 x 10(-9) M and 2.2 x 10(-9) M, respectively). The relationship between the structure of eglin c and the inhibitory activity against the above enzymes is also described.
Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.
A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).
Collagen fibres form the stable architecture of connective tissues and their breakdown is a key irreversible step in many pathological conditions. The destruction of collagen is usually initiated by proteinases, the best known of which is the metalloproteinase collagenase (EC 3.4.24). Collagenase and related metalloproteinases are regulated at the level of their synthesis and secretion, through the action of specific stimuli such as hormones and cytokines, and also at the level of their extracellular activity through the action of a specific inhibitor, TIMP (tissue inhibitor of metalloproteinases), which irreversibly forms inactive complexes with metalloproteinases. Although the mechanisms governing the production of TIMP are unknown, immunologically identical forms of this glycoprotein have been detected in a wide variety of human body fluids and cell and tissue culture media. We therefore suggested that under physiological conditions this ubiquitous inhibitor predominates over active metalloproteinases and that tissue destruction may arise when any perturbation of this controlling excess arises. However, further progress towards testing this theory has been hindered by a lack of knowledge about the structure of TIMP and insufficient material for studying it in model systems. Here we describe the structure of TIMP predicted from its complementary DNA, its synthesis in Escherichia coli and transfected animal cells, and the finding that it is identical to a protein recently reported to have erythroid-potentiating activity (EPA).
A new in vitro cell system, Line human endometrial cancer-one (HEC-1), derived from adenocarcinoma of human endometrium has been established and has successfully proved stable proliferation in continuous tissue culture since May, 1968. The cytologic findings of HEC-1 reveal such anaplastic features as anisonucleosis, nucleolar pleomorphism, and piling-up tendency in cellular arrangement. Distribution of the chromosome number is found at the diploid range, and the apparent marker chromosome has been identified without exception. When transplanted into a hamster cheek pouch, HEC-1 demonstrates a definite tumor formation whose histologic picture reveals papillary adenocarcinoma. The original characteristics of endometrial cancer have been precisely maintained in this in vitro culture system.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Tumour cells traverse epithelial and endothelial basement membranes during the successive stages of the metastatic process. At the transition from in situ to invasive carcinoma, local dissolution of the basement membrane is observed microscopically1,2, and coincides with tumour cell invasion of the underlying stroma. Tumour cells further traverse the endothelial basement membrane during entry into and egress from blood vessels3-5. Electron microscopic studies have shown local dissolution of basement membrane at its area of contact with invading tumour cells, suggesting an enzymatic mechanism3,6,7. Basement membranes are resilient structures which present a mechanical barrier to invasion8. Type IV collagen is a major structural protein of basement membranes and is chemically and genetically distinct from stroma collagen types I and III and cartilage collagen type II9,10. Previously characterised animal collagenases which cleave collagen types I; II and III fail to degrade type IV collagen11,12. We have recently purified about 1,000-fold and characterised a neutral protease activity preferential for type IV collagen from metastatic tumour cells and shown that it (1) produces specific degradation products, (2) has a molecular weight of 65,000, (3) is not plasmin or a cathepsin, by pH and inhibitor studies, and (4) does not significantly degrade other collagens or fibronectin12,13. Here we extend the relevance of this finding by quantitating the ability of several murine tumour cell lines of known metastatic potential to degrade type IV collagen. The cell lines with the highest incidence of spontaneous metastasis exhibit the greatest level of type IV collagen-degrading activity in two different assays using either living cells or media obtained from cell cultures.
Highly purified antibodies to two ubiquitous components of basement membrane, type IV collagen and laminin, were applied to both fresh-frozen and formalin-fixed tissue sections of a variety of invasive carcinomas, carcinomas in situ, and their "look-alike" benign counterparts. These included lesions of the breast (infiltrating ductal carcinoma, comedocarcinoma, and sclerosing adenosis); lesions of the skin (squamous cell carcinoma, Bowen's disease, and pseudoepitheliomatous hyperplasia); lesions of the pancreas (adenocarcinoma and pancreatitis); lesions of the prostate (adenocarcinoma and benign prostatic hyperplasia); and other epithelial lesions of the invasive, in situ, and benign category. By both immunofluorescence and immunoperoxidase techniques, benign and in situ lesions showed intact basement membranes with linear staining of type IV collagen and laminin. The majority of invasive carcinomas, in contrast, lacked immunoreactivity for both of these basement membrane components. In cases of in situ carcinoma with microinvasion, there was thinning, fragmentation, and disruption of the basement membrane in the foci of microinvasion but not elsewhere. Utilizing antibodies to type IV collagen and laminin aids in both understanding the pathophysiology of the invasive process and the recognition of its presence in tissue sections.
Signaling by tyrosine kinase receptors is mediated by selective interactions between individual Src homology 2 (SH2) domains of cytoplasmic effectors and specific phosphotyrosine residues in the activated receptor. Here, we report the existence in the hepatocyte growth factor/scatter factor (HGF/SF) receptor of a multifunctional docking site made of the tandemly arranged degenerate sequence YVH/NV. Phosphorylation of this site mediates intermediate- to high-affinity interactions with multiple SH2-containing signal transducers, including phosphatidylinositol 3-kinase, phospholipase C gamma, pp60c-src, and the GRB-2-Sos complex. Mutation of the two tyrosines results in loss of biological function, as shown by abrogation of the transforming activity in the oncogenic counterpart of the receptor. The same bidentate motif is conserved in the evolutionarily related receptors Sea and Ron, suggesting that in all members of the HGF/SF receptor family, signal transduction is channeled through a multifunctional binding site.
Pancreatic cancer shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissue (collagens type I and III, fibronectin). In this study we have analyzed the balance of expression of mRNAs encoding extracellular matrix components (collagens I, III and IV, laminin, fibronectin), extracellular matrix-degrading metalloproteinases (MMP-1, -2, -3 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in pancreatic cancer and control pancreatic tissue by Northern-blot analysis and mRNA in situ hybridization. Transcripts for MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were not detectable in pancreatic cancer and control tissues. Steady-state levels of transcripts encoding extracellular matrix proteins, MMP-2 (72-kDa collagenase IV), MMP-9 (92-kDa collagenase type IV), TIMP-1 and TIMP-2 were elevated in the majority of pancreatic-cancer tissue samples as compared to control pancreatic tissue. A good correlation was seen between overexpression of these MMPs and TIMPs and the steady-state levels of transcripts coding for extracellular matrix proteins, the amount of collagen protein and the severity of the desmoplastic reaction. In situ hybridization studies localized transcripts coding for collagens type I and III to spindle-shaped stromal cells, whereas transcripts for MMP-2, MMP-9, TIMP-1 and TIMP-2 were found in both stromal and tumor cells. However, MMP-2 transcripts appeared to be more abundant in stromal cells, TIMP-1 and TIMP-2 transcripts were evenly distributed over tumor and stromal cells and relatively more MMP-9 transcripts were found in tumor cells. We conclude that, in human pancreatic cancer, MMP-2, MMP-9, TIMP-1 and TIMP-2 may be involved in processes leading to the strong desmoplastic reaction observed in these tumors. Both stromal and tumor cells appear to be the source of MMPs and TIMPs in human pancreatic cancer.
We examined the in situ distribution of basement membrane collagen (Col IV), matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) by immunohistochemistry and their mRNA levels by Northern blot analysis in 14 cases of squamous cell carcinoma of the lung. Elevated mRNA levels of MMP-2 and Col IV were demonstrated in all the cases examined and were associated with in situ disruption of basement membranes around the tumor nests. In contrast, TIMP-1 mRNA levels were not altered. MMP-2, MMP-9 and TIMP-1 were localized in tumor cells, stromal fibroblasts and endothelial cells. There were no significant correlations between these parameters and clinical staging. The results suggest that the degrading enzymes of basement membrane collagen play an important role in the invasion and metastasis of human squamous cell carcinoma of the lung.
Our purpose was to evaluate type IV collagenase in ovarian and endometrial cancer tissues.
Tissue specimens were obtained from patients with ovarian and endometrial cancer and uterine myoma. Gelatinase activity was detected by zymography and quantitated by densitometer.
Four dominant gelatinases were detected in ovarian and endometrial cancer tissues: 200, 130, 92, and 72 kd gelatinase. Other forms observed were 83 kd gelatinase, which is an active form of 92 kd gelatinase, and 66 kd gelatinase, which is an active form of 72 kd gelatinase. Densitometric analysis showed that the 92 kd/72 kd ratio in ovarian cancer tissues was significantly higher than in normal ovarian tissues (p < 0.05) and that the 66 kd/72 kd ratio was higher in ovarian cancer tissues (p = 0.07). Both ratios in endometrial cancer tissues were significantly higher than in normal endometrial tissues (p < 0.05).
Gelatinase activity was remarkably higher in ovarian and endometrial cancer tissues. Especially, 92, 83, and 66 kd gelatinases were clearly detected in cancer tissues, suggesting that these gelatinases were related to the malignant phenotype, because degradation of the components of the basement membrane such as type IV collagen is necessary for cancer cells to metastasize.
Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr⁸⁵ N terminus; Mr 48,000). MT1-MMP activated procollagenase-3 via a Mr 56,000 intermediate (Ile³⁶ N terminus) to 48,000 which was the result of the cleavage of the Glu⁸⁴-Tyr⁸⁵ peptide bond. We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family.
In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys³⁸-Glu³⁹ and Arg⁷⁶-Cys⁷⁷ peptide bonds in the propeptide domain. Autoproteolysis then resulted in the release of the rest of the propeptide domain generating Tyr⁸⁵ N-terminal active collagenase-3. However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity.
Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a Mr 56,000 intermediate form to the final Mr 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation. We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin A-stimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.
Substantial evidence indicates that proteolytic degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Our previous work has demonstrated elevated secretion by cultured ovarian adenocarcinoma cells of two gelatinolytic metalloproteinases, a 72-kDa enzyme resembling matrix metalloproteinase 2 (MMP-2) and a 92-kDa enzyme resembling MMP-9 (Moser et al, Int. J. Cancer 56, 552-559, 1994). To assess the potential in vivo relevance of these enzymes, we have examined ovarian carcinoma ascites using gelatin substrate zymography. MMP species identical to those secreted from several well-characterized ovarian adenocarcinoma cell lines were found in the majority of ascites: MMP-2-like gelatinase (23 of 23 cases) and MMP-9-like gelatinase (18 of 23 cases), suggesting a prevalence of these species in the ovarian carcinoma microenvironment and their availability for tumor-associated proteolysis. The contribution of these proteinases to ovarian cancer invasion was further demonstrated by experiments measuring tumor cell-mediated proteolysis of native endothelial cell extracellular matrix (ECM) and tumor cell invasion of reconstituted basement membrane (Matrigel). These data showed that secretion of type IV collagenase activity by a series of independently isolated ovarian adenocarcinoma cell lines correlated well with the ability of these cells to proteolyze the ECM and invade the basement membrane. Furthermore, we have identified and characterized an ovarian carcinoma-associated gelatinase, the 72-kDa MMP found in conditioned media of the DOV 13 cell line, as MMP-2. This enzyme was identical to the previously described MMP-2 from other sources by Western blot, amino terminal sequence, and substrate specificity. Additionally, a large portion of the MMP-2 activity found in DOV 13 conditioned media is active without organomercurial treatment, suggesting that ovarian cancer cells have an endogenous activator of the zymogen. Together, these data suggest that ECM proteolysis mediated by tumor-associated proteinases plays an important role in the invasion and/or metastasis of ovarian carcinoma.
The expression of MMP-2, MMP-9, TIMP-1, TIMP-2, and the urokinase receptor were examined in fetal and normal prostate tissues, benign prostatic hyperplasia and prostate cancer (n = 117). In situ hybridization with digoxigenin-labeled oligonucleotide probes demonstrated that TIMP-1 and TIMP-2 were expressed at elevated levels in the stroma of Gleason sum 5 tissues, whereas MMP-2 and MMP-9 were expressed at relatively low levels. In higher Gleason sum tissues (GS 8-10), TIMP-1 and TIMP-2 were not expressed, whereas MMP-2 and MMP-9 were intensely expressed. Furthermore, TIMP-1 and TIMP-2 expression was high in organ-confined specimens (OC, n = 43), somewhat lower in specimens with capsular penetration (CP, n = 29), and low or negative in samples with surgical margin/seminal vesicle (M/SV, n = 17) and lymph node (LN, n = 13) involvement. In contrast, MMP-2 and MMP-9 expression was low in the OC tissues; and noticeably higher in CP, M/SV, and LN specimens. Finally, correlation of TIMP and MMP expression with GS and pathological stage versus cure rate further revealed that a high percentage of organ-confined, GS 5 specimens expressing TIMP and little MMP were cured. In comparison, few of the GS 7-10 patients with capsular penetration and expressing MMP and little TIMP were cured. The data suggest that TIMP-1 (and TIMP-2) and MMP-2 (and MMP-9) are independent predictors of outcome.
Receptor tyrosine kinases are key regulators of cellular function including cell growth, differentiation, migration, and morphogenesis. Disruptions of receptor tyrosine kinase signaling pathways are often associated with changes in cellular proliferative capacity and tumorigenesis. Both receptor-specific and cell type-specific factors may contribute to the ultimate cellular responses observed after receptor activation. In this regard, we find that both normal keratinocytes and their tumorigenic counterparts display differential responses to activation of receptor tyrosine kinases. Multiple ligands were mitogenic for keratinocytes, but only epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and scatter factor/hepatocyte growth factor (SF/HGF) promoted cell motility as assessed by colony dispersion (scattering) and in vitro reepithelialization. Interestingly, growth factor specificity for motility coincided with ligand-mediated cell invasion through a reconstituted basement membrane and induction of the 92-kDa metalloproteinase (MMP-9) activity as determined by gelatin zymogram analysis. Inhibitors of MMP activity or addition of an MMP-9 neutralizing antibody resulted in the loss of growth factor-induced colony dispersion, suggesting a functional role for MMP-9 induction during this response. Coordinate regulation of MMP-9 induction and the migratory response are likely to contribute to the enhanced invasive potential observed in response to EGF and SF/HGF. Our findings suggest that alternate receptor-mediated signaling pathways leading to differences in gene expression may be involved in complex cellular responses such as colony dispersion or invasion.
The overall goal of this study was to investigate the role of the hepatocyte growth factor (HGF)/Met pathway in the pathophysiology of invasive endometrial carcinoma. Our objectives were (1) to examine expression of HGF and Met in surgical endometrial carcinoma specimens and endometrial carcinoma cell lines, and (2) to determine if HGF would stimulate invasion of endometrial carcinoma cell lines in vitro.
Using RT-PCR and Western immunoblotting, endometrial carcinoma specimens and the endometrial carcinoma cell lines KLE, HEC-1A, HEC-1B, and RL-95 were examined for expression of HGF and Met. A Boyden chamber invasion assay using collagen type I coated 8-microm porous membranes was then used to determine if HGF would stimulate cell invasion. Last, we assessed the capacity of endometrial stromal cells, isolated from normal human endometrium, to produce HGF as determined by an enzyme-linked immunosorbent assay and to stimulate invasion of the KLE cell line.
All of the endometrial carcinoma tissue samples were found to express Met mRNA, and two of four samples expressed HGF mRNA. However, the endometrial carcinoma cell lines expressed only Met and not HGF mRNA. Both the endometrial carcinoma tissue specimens and the endometrial carcinoma cell lines expressed the 140-kDa Met protein. HGF induced the invasion of the KLE and HEC-1A cells through the collagen-coated membranes in a dose-dependent fashion. The optimal concentration of HGF was between 10 and 100 ng/ml. HGF (10 ng/ml) stimulated KLE invasion 1.8-fold (P < 0.05) and HEC-1A invasion 6.5-fold (P < 0.05). During exposure to endometrial stromal cell conditioned medium containing HGF as determined by ELISA, invasion of the KLE cell line was stimulated 2.5-fold (P < 0.05).
These results demonstrate that HGF stimulates the invasion of endometrial carcinoma cells in vitro. Since endometrial adenocarcinoma specimens express Met, these findings suggest that the HGF/Met pathway may play a role in the invasive progression of endometrial carcinoma.