Peritumoral Fibroblasts Research Articles

MicroRNA-21 in Pancreatic Ductal Adenocarcinoma Tumor-Associated Fibroblasts Promotes Metastasis

IntroductionPancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.MethodsIn-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.ResultsmiR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing α-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.ConclusionsmiR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.

EMMPRIN expression positively correlates with WHO grades of astrocytomas and meningiomas

High-grade primary brain tumors possessed poor outcome due to invasiveness. Extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates peri-tumoral fibroblasts to secrete matrix metalloproteinase and promote invasiveness. This study hypothesized that high-grade brain tumors overexpress EMMPRIN. Analyzing the public delinked database from the Gene Expression Omnibus profile, the results showed that the EMMPRIN mRNA level was higher in WHO grade IV (n = 81) than in grade III (n = 19, p < 0.0005) astrocytomas and non-tumor brain tissue controls (n = 23, p < 0.00001). The results of tissue microarray-based immunohistochemical (IHC) staining revealed that EMMPRIN levels positively correlated with WHO grades for astrocytomas (p = 0.008) and meningiomas (p = 0.048). EMMPRIN mRNA levels in conventional glioma cell lines (n = 36) was not less than those in glioma primary culture cells (n = 27) and glioblastoma stem-like cells (n = 12). The GBM8401, U87MG, and LN229 human glioma cell lines also overexpressed EMMPRIN. Hematoxylin and eosin, IHC, and immunofluorescence staining of xenografts confirmed that high-grade brain tumors overexpressed EMMPRIN. Lastly, Kaplan–Meier analysis revealed poorer survival in WHO grade IV (n = 56) than in grade III astrocytomas (n = 21, by log-rank test; p = 0.0001, 95 % CI: 1.842–3.053). However, in high-grade astrocytomas, there was no difference in survival between high and low EMMPRIN mRNA levels. Thus, this study identified that high-grade brain tumors overexpress EMMPRIN, which positively correlates with WHO grades in human astrocytomas and meningiomas, and suggests that EMMPRIN may be a therapeutic target of brain tumor.

P4-18-01: Prognostic Role of Tumor and Stromal p16/INK4A in Ductal Carcinoma In Situ (DCIS).

Abstract Background DCIS is a pre-invasive breast disease diagnosed in ∼50,000 women/year in the US. The standard treatment recommendation is for mastectomy or breast-conserving surgery plus radiotherapy (BCS+RT). However, only ∼10% of BCS patients avoid recurrence due to RT, and RT provides little or no survival benefit. Thus, it is important to stratify patients by risk to help tailor treatment to maximize benefit and minimize unnecessary adverse effects and costs. Material and Methods: Tissue microarrays that included tumor cores from all women diagnosed with pure DCIS between 1986 and 2004 in Uppland and Västmanland, Sweden (n=458) were stained and scored for p16/INK4A (clone E6H4) using immunohistochemistry (IHC). Semi-quantitative manual scoring was done separately in the tumor epithelial cells and peri-tumoral carcinoma-associated fibroblasts (CAFs). Based on previous reports, thresholds to define low and high p16 levels were set at 25% and 5% positive staining, respectively, for the tumor and stromal components. HER2 IHC scoring was reported previously. Clinicopathologic characteristics of the set of patients for which complete marker data was obtained (n=176) were not statistically different than the full set of patients. For survival analysis, local ipsilateral DCIS and invasive recurrences, as well as lymph node and distant metastases, were included as events (patients were censored if they had contralateral breast or other cancers as the first event). Results: Patients were stratified into low and high risk groups using a multivariate model incorporating tumor p16, stromal p16, HER2, and margin status. Patients treated with BCS only (n=67) had a baseline 10-year recurrence rate of 31%. The model identified high and low risk groups with 10-year recurrence rates of 38% and 7%, respectively (hazard ratio=7.1, p=0.009). Similar results were achieved when BCS only and BCS+RT patients were combined (n=130). Interestingly, all six recurrence events in the set of patients who received full mastectomies (n=43) were in the model high risk group. Discussion: Previous studies have shown mixed results when p16 was used as a single marker. Our results suggest a prognostic interaction between tumor and stromal p16, as well as HER2, that will form the basis of a more comprehensive multi-marker IHC-based prognostic panel to assess risk of recurrence in DCIS patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-18-01.

Fibrous Stroma Is Associated with Poorer Prognosis in Lung Squamous Cell Carcinoma Patients

Cancer tissue is composed of various stromal cells forming cancer-specific microenvironments. Peritumoral stroma is reportedly composed of activated fibroblasts that can influence the biological properties of tumor cells, mainly their local aggressiveness and their ability. The aim of this study was to examine whether the histological properties of peritumoral stroma are correlated with squamous cell carcinoma (SqCC) aggressiveness and clinical outcome. A series of 220 pathological stage I lung SqCC were categorized into two types according to the histological properties of the peritumoral stroma, "fibrous stroma type" (n = 85), and "thin stroma type" (n = 135), and compared the prognostic significance. Furthermore, we compared the immunohistochemical properties of the SqCC cells surrounded by "fibrous stroma" with those of the SqCC cells surrounded by "thin stroma." The prognosis of the patients with fibrous stroma-type tumors was significantly poorer than that of the thin stroma type with regard to both recurrence-free survival (p = 0.005) and overall survival (p = 0.008). A multivariate analysis showed that the presence of a fibrous stroma was an independent prognostic factor (p = 0.030). Compared with the SqCC cells with a thin stroma, the SqCC cells with a fibrous stroma exhibited reduced expression of E-cadherin (55.9 versus 126.0, p < 0.001) and an increased expression of laminin-5γ2 (94.6 versus 25.0, p = 0.001), matrix metalloproteinase-7 (26.0 versus 3.50, p = 0.009), and c-Met (64.0 versus 36.5, p = 0.033). SqCC with a fibrous stroma displayed higher invasive phenotype and were associated with a significantly poor prognosis. The current results suggest that the microenvironment created by both SqCC cells and the peritumoral fibroblasts may facilitate cancer aggressiveness.

Tumor-secreted lysophostatidic acid accelerates hepatocellular carcinoma progression by promoting differentiation of peritumoral fibroblasts in myofibroblasts

Hepatocellular carcinoma (HCC) occurs in fibrotic liver as a consequence of underlying cirrhosis. The goal of this study was to investigate how the interaction between HCC cells and stromal fibroblasts affects tumor progression. We isolated and characterized carcinoma-associated fibroblasts (CAFs) and paired peritumoral tissue fibroblasts (PTFs) from 10 different patients with HCC and performed coculture experiments. We demonstrated a paracrine mechanism whereby HCC cells secrete lysophostatidic acid (LPA), which promotes transdifferentiation of PTFs to a CAF-like myofibroblastic phenotype. This effect is mediated by up-regulation of specific genes related to a myo/contractile phenotype. After transdifferentiation, PTFs expressed α-smooth muscle actin (α-SMA) and enhanced proliferation, migration, and invasion of HCC cells occur. A pan-LPA inhibitor (α-bromomethylene phosphonate [BrP]-LPA), or autotaxin gene silencing, inhibited this PTF transdifferentiation and the consequent enhanced proliferation, migration, and invasion of HCC cells. In vivo, PTFs coinjected with HCC cells underwent transdifferentiation and promoted tumor progression. Treatment with BrP-LPA blocked transdifferentiation of PTFs, down-regulated myofibroblast-related genes, and slowed HCC growth and progression. Patients with larger and metastatic HCC and shorter survival displayed higher serum levels of LPA. Analysis of microdissected tissues indicated that stroma is the main target of the LPA paracrine loop in HCC. As a consequence, α-SMA-positive cells were more widespread in tumoral compared with paired peritumoral stroma. Our data indicate that LPA accelerates HCC progression by recruiting PTFs and promoting their transdifferentiation into myofibroblasts. Inhibition of LPA could prove effective in blocking transdifferentiation of myofibroblasts and tumor progression.

Increased expression of ADAM12 and ADAM17 genes in laser-capture microdissected breast cancers and correlations with clinical and pathological characteristics

ADAMs (a desintegrin and metalloprotease) are transmembrane glycoproteins involved in cell growth, differentiation, motility, and respectively, tumor growth and progression. Our aim was to evaluate ADAM12 spliced variants (ADAM12L – long membrane-bound and ADAM12S – secreted-short variant) and ADAM17 genes expression in breast cancers and to correlate their level of expression with clinical and pathological characteristics.Expression of ADAMs was analyzed using quantitative reverse-transcription polymerase chain reaction in laser-capture microdissected specimens of breast cancers and corresponding non-neoplastic breast tissues from 92 patients. The proteins’ expression was confirmed by immunohistochemistry. Significantly elevated amounts of ADAM12L, ADAM12S and ADAM17 transcripts were found in malignant breast cells compared with normal breast tissue and both ADAMs proteins showed moderate to strong immunoexpression in tumor cells and peritumoral fibroblasts. ADAM12L and ADAM12S expressions were correlated with age, younger patients having higher expression of ADAM12L and ADAM12S; ductal cancers had higher expression of ADAM12L compared with lobular types, whereas ADAM12S was higher expressed in lobular cancers; higher expressions were found for both ADAM12 and ADAM17 in HER2/neu positive and highly proliferative cancers. High-grade cancers showed significantly increased expression of ADAM17. Our study on laser-capture microdissected specimens confers motivation for future work on development of ADAM-selective inhibitors for treatment of breast cancers.

Abstract 511: Adipocyte-derived fibroblasts contribute to the desmoplastic reaction in breast cancer: A new link between breast cancer and obesity

Abstract Now, it is widely established that malignant tumors, not merely grow into a preexisting interstitial tissue, but they actively form a new stroma and modify their composition. Accordingly, one of the features of many carcinomas, particularly breast, is the presence of a dense collagenous stroma, the so-called desmoplastic response, comprising fibroblast-like cells which play a crucial role in tumor progression. Peritumoral fibroblasts are composed of several subpopulations that are morphologically undistinguishable and their origins remain debated. Our results suggest that a part of this fibroblast could arise from the “reorientation” of mature adipocytes, highly represented in breast tumour microenvironment. Using an original 2D coculture system, we show that mature adipocytes cocultivated with breast tumour cells for 3 to 8 days lose their lipid content as well as their adipocyte differentiation markers (HSL, Adipsin, Adiponectin, C/EBPα, PPARγ, Resistin). This process is associated with intense morphological and functional changes leading to an activated fibroblast phenotype (exhibition of actin stress fibers, increase of focal adhesion sites). In this way, we have identified for the first time a new fibroblastic population called Adipocyte Derived Fibroblast (ADF). Interestingly, the reorientation occur in vivo since the injection of far red fluorescent tumour cells into a mice fatpad containing GFP-expressing adipocytes induce the apparition of GFP-fibroblast-like cells that interdigitate with tumour cells. Moreover, our results show that the ADF phenotype depends on the reactivation of the Wnt/β-catenin pathway. Finally, ADFs exhibit an increase of migratory and invasive abilities, with enhanced profibrotic secretions (fibronectin and collagen 1) suggesting that these cells could be key players in desmoplastic reaction. Ongoing experiments are performed to assess the presence of ADFs in breast human tumors. Our results might provide an explanation for the poor prognosis observed in localized breast cancer in obese women, since the nature of the desmoplastic reaction and the secretion pattern of ADF might be profoundly altered in this physiopathogical condition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2011-511

Fibroblast-activation protein: a single marker that confidently differentiates morpheaform/infiltrative basal cell carcinoma from desmoplastic trichoepithelioma

Microscopically, differentiating desmoplastic trichoepithelioma from morpheaform/infiltrative basal cell carcinoma can be difficult as both show ‘islands and strands of basaloid cells embedded in a sclerotic stroma’. A superficial shave biopsy further compounds the diagnostic conundrum. Although a plethora of immunohistochemical markers have been touted as being of use as adjunct histologic tools, none thus far appears to be consistent and reliable in terms of specificity and/or sensitivity. Fibroblast-activation protein, a type II membrane-bound glycoprotein belonging to the serine protease family, is expressed in the granulation tissue of healing wounds. More recently, it has been identified as a marker of reactive tumor stromal fibroblasts, as it is reportedly selectively expressed in peritumoral stromal fibroblasts of multiple epithelial cancers including cutaneous malignancies such as basal cell carcinoma. Given this, we sought to ascertain the use of fibroblast-activation protein in distinguishing morpheaform/infiltrative basal cell carcinoma from desmoplastic trichoepithelioma. Immunohistochemical staining for fibroblast-activation protein was performed on desmoplastic trichoepithelioma (n=25) and morpheaform/infiltrative basal cell carcinoma (n=25), with the control group comprising scars from reexcision specimens (n=10). As expected, fibroblast-activation protein expression was observed in stromal fibroblasts of all control cases (10 of 10, 100%). Of interest, fibroblast-activation protein expression was observed in peritumoral fibroblasts of all cases of morpheaform/infiltrative basal cell carcinoma (25 of 25, 100%) but not in any cases of desmoplastic trichoepithelioma (0 of 25, 0%). A gradient of fibroblast-activation protein expression was observed in morpheaform/infiltrative basal cell carcinoma with more intense expression noted in fibroblasts abutting the tumor cells, a less intense expression in the distal peritumoral stromal portion, and minimal to loss of expression in adjacent normal tissue. In summary, findings from this study underscore the use of fibroblast-activation protein as a histologic adjunct in confidently differentiating morpheaform/infiltrative basal cell carcinoma from desmoplastic trichoepithelioma.

CD10‐bearing fibroblast inhibits matrigel invasive potency of interleukin‐1α‐producing squamous cell carcinoma by diminishing substance P levels in the tumor microenvironment

CD10 is a neutral endopeptidase, which cleaves various peptide substrates including substance P. CD10 expression has been detected in peritumoral fibroblasts (Fb) within the invasive area of various cancers such as squamous cell carcinoma (SCC). However, the biological significance of CD10-bearing Fb remains largely unknown. We examined dynamic interactions of Fb with tumorigenic A431 SCC cells or non-tumorigenic HaCaT squamous cells. The SCC and HaCaT cells did not synthesize CD10, while Fb constitutively expressed CD10. When co-cultured, SCC markedly upregulated fibroblastic CD10 expression compared with HaCaT, which was mainly attributable to SCC-derived interleukin-1α (IL-1α). Both SCC and Fb autonomously secreted substance P, which eventually enhanced the invasive capacity of SCC in a matrigel invasion assay by upregulating matrix metalloproteinase (MMP)-1 and MMP-2, but not MMP-9. Transfection of siRNA for CD10 successfully knocked down the CD10 expression in Fb (CD10ND-Fb). In the presence of CD10ND-Fb, substance P levels in supernatants as well as MMP production and the invasive potency of SCC were significantly augmented compared with control scramble RNA-transfected Fb. We also transfected CD10 vector to Fb and found that the matrigel invasive ability of SCC cells was downregulated co-cultured with CD10 vector-transfected Fb rather than empty vector-transfected Fb. In conclusion, the CD10-bearing Fb generated by SCC-derived IL-1 inhibited the invasive capacity of SCC by diminishing the microenvironmental concentration of substance P.

Cathepsin K-upregulation in fibroblasts promotes matrigel invasive ability of squamous cell carcinoma cells via tumor-derived IL-1α

Cathepsin K (CTSK), a cysteine protease with strong collagenolytic properties, is involved in extracellular matrix turnover. In the previous studies, CTSK expression was detected in peritumoral fibroblasts (Fbs) around squamous cell carcinoma (SCC), but not in those surrounding benign epidermal tumors. However, the mechanism governing CTSK expression in epidermal tumors remains unclear. To study the regulatory mechanisms of fibroblastic CTSK expression in the SCC-stromal interaction. We examined dynamic interactions of Fbs with tumorigenic SCC cells (A431 and A253) or normal human keratinocytes. SCC cells and normal keratinocytes did not synthesize CTSK, while Fbs constitutively expressed CTSK. When cocultured, SCC cells upregulated fibroblastic CTSK expression more potently than did normal keratinocytes, which was mainly attributable to SCC-derived IL-1α. Coculturing Fbs with SCC cells significantly augmented the matrigel invasive ability of SCC cells, which was downregulated when cocultured with CTSK knockdown Fbs or in the presence of neutralizing anti-IL-1α antibody. The CTSK-upregulated Fbs generated by SCC-derived IL-1α may play a crucial role in the progression and invasion of SCC.

Pathology of soft‐tissue tumors: Daily diagnosis, molecular cytogenetics and experimental approach

This article reviews problems in diagnostic pathology and molecular cytogenetics of soft-tissue tumors. Also discussed are the origin of soft-tissue sarcomas and the molecular basis of effective target therapy for sarcomas. Molecular cytogenetic analysis of tumor-specific chromosomal translocations and associated fusion gene transcripts offers a useful adjunct to the diagnosis of soft-tissue tumors, but recent studies have indicated a growing number of fusion gene variations in each tumor type. In pleomorphic sarcoma/malignant fibrous histiocytoma, the alternative lengthening of telomeres (ALT) mechanism may result in formation of anaphase bridges and marked nuclear pleomorphism. The histogenesis of soft-tissue sarcomas has been a matter of controversy. In the present experimental model using s.c. injection of 3-methylcholanthrene in C57BL/6 mice pretreated with bone marrow-transplantation from green fluorescent protein (GFP)-positive green mice, the bone marrow-derived mesenchymal stem cells as well as the tissue-resident mesenchymal cells in the peripheral soft tissues are possible originators of sarcomagenesis. Little is known about a molecular basis of target therapy for sarcomas. Platelet-derived growth factor-BB (PDGF-BB) enhances the invasive activity of malignant peripheral nerve sheath tumor (MPNST) cells through platelet-derived growth factor receptor (PDGFR) phosphorylation, whereas imatinib mesylate inhibited such activity, suggesting that targeting PDGFR-beta may result in the establishment of novel treatment for MPNST. In addition, emmprin is a transmembrane glycoprotein on tumor cells that stimulates peritumoral fibroblasts to produce matrix metalloproteinases (MMP), playing a crucial role in tumor progression, invasion and metastasis. The MMP upregulation mechanism mediated by tumor-associated emmprin may be a potentially useful target in anti-tumor invasion therapy for sarcomas.

Expression of extracellular matrix metalloproteinase inducer and enhancement of the production of matrix metalloproteinase-1 in tongue squamous cell carcinoma

Recent studies have found that in addition to promoting cellular invasion, overexpression of metalloproteinase -1 (MMP-1) is associated with the initial stages of cancer development. Extracellular matrix metalloproteinase inducer (EMMPRIN), a transmembrane glycoprotein, has been reported to be highly expressed in tumor cells and induce production of MMPs from peritumor fibroblasts (PTFs) adjacent to the tumor cells. The expression of EMMPRIN in tongue squamous cell carcinoma (SCC) was investigated in this study. It was found that EMMPRIN was expressed at the cell membrane throughout the entire lesion in tongue SCC. Immunofluorescence staining localized EMMPRIN to the cell membrane in a highly invasive tongue SCC cell line (Tca 8113). EMMPRIN mRNA was expressed at a high level in Tca 8113, whereas MMP-1 mRNA was expressed in PTF but harder to be detected in Tca 8113. Co-culture of Tca 8113 with PTF stimulated production of MMP-1. EMMPRIN was highly expressed in tongue SCC, and could induce local production of MMP-1. These data indicate that EMMPRIN might play an important role in tongue SCC progression and invasion.

Expression of the metalloproteases MMP-1, MMP-2, MMP-3, MMP-9, MMP-11, TIMP-1 and TIMP-2 in angiocentric midfacial lymphomas

BackgroundExtranodal T/NK cell lymphomas possess distinctive clinico-pathological characteristics: they are angiocentric, exhibit extensive necrosis. Prognosis is poor in the short term. The objective is to explore the expression of different MMPs in the cells and stroma which are around of the blood vessels damaged and their correlation with clinico-pathological parameters.Patients and methodsTwenty cases of this type of lymphomas were studied and collected patient clinical data. The expressions of MMP-1, 2, 3, 9, 11, 13 and TIMP-1, 2 were studied by immunohistochemistry. Ultrastructural studies were performed in two cases. Statistical analysis was done with Fisher's exact test, Chi2 test.ResultsOf the 20 patients, 13 were men with median age of 43 years. In 13 patients the primary tumor was localized in the nasal cavity. Treatment was combined chemotherapy and radiotherapy in 60%. The 55% advanced clinical stages, 70% died from the disease. There were neoplastic cell and peritumoral fibroblasts positivity to MMP-1 and MMP-11 in most of the cases. The MMPs-2, 3 and 9 were expressed in neoplastic cell between 30 to 65%of the cases. TIMP-1 was presented mainly in the epithelium and TIMP-2 was poor expressed of the all cases.ConclusionThere were no statistical significance between the different enzymes used and the clinical parameters, besides status and survival of the patients. It is necessary to study more enzymes and focus them to quantify and determine their activity, in order to have a better correlation with histological features in this type of neoplasm.

Relationship between the expression of matrix metalloproteinase-13 protein and other biomarkers, prognosis in invasive breast cancer

The study was designed to investigate the expression patterns of metalloproteinase (MMP)-13 protein in invasive breast carcinoma and to determine the clinicopathological and prognostic values of its various localization and relation to the tumor phenotypes. Immunohistochemistry was performed on paraffin-embedded tissue array from 263 invasive breast carcinomas to investigate the protein expressions of MMP-13, estrogen receptor, progesterone receptor, HER2, MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinases (TIMP)-1, TIMP-2. MMP-13 protein was detected in the cytoplasm of carcinoma cells and peritumoral fibroblasts. High level expression of MMP-13 protein in tumor cells was associated with more lymph node involvement and higher tumor grade (both P < 0.01), and positively correlated with HER2 (P = 0.015) and TIMP-1 protein (P < 0.01) expression in carcinoma cells. Moreover, high expression of MMP-13 was associated with shortened overall survival for the entire patient population and the patient group with positive lymph node. Tumor cell derived MMP-13 had different impact on patients with different HER2 status. Peritumoral fibroblasts derived MMP-13 protein, although correlated with tumor cell derived MMP-13 and associated with lymph node stage and HER2 expression, was found having less prognostic impact. Univariate survival analysis showed that the tumor size, grade, lymph node status, PR status, HER2 expression, tumors TIMP-1 and MMP-13 expression were prognostic factors. However, multivariate survival analysis showed that only tumor size, lymph node status, HER2 expression, tumors TIMP-1 and MMP-13 were independent prognostic factors. MMP-13 protein expressed by tumor cells correlates with the invasion and metastasis of breast carcinoma, and therefore, may serve as a poor prognostic marker for the patient.

Tumor-derived matrix metalloproteinase-13 (MMP-13) correlates with poor prognosis of invasive breast cancer

BackgroundExperimental evidence suggests that matrix metalloproteinase-13 (MMP-13) protein may promote breast tumor progression. However, its relevance to the progression of human breast cancer is yet to be established. Furthermore, it is not clear whether MMP-13 can be used as an independent breast cancer biomarker. This study was conducted to assess the expression profile of MMP-13 protein in invasive breast carcinomas to determine its diagnostic and prognostic significance, as well as its correlation with other biomarkers including estrogen receptor (ER), progesterone receptor (PR), Her-2/neu, MMP-2, MMP-9, tissue inhibitor of MMP-1 and -2 (TIMP-1 and TIMP-2).MethodsImmunohistochemistry (IHC) was performed on paraffin-embedded tissue microarray containing specimens from 263 breast carcinomas. The intensity and the extent of IHC were scored by pathologists in blind fashion. The correlation of the gene expression profiles with patients' clinicopathological features and clinical outcomes were analyzed for statistical significance.ResultsMMP-13 protein was detected in the cytoplasm of the malignant cells and the peritumoral stromal cells. MMP-13 expression by tumor cells (p < 0.001) and stromal fibroblasts (p <0.001) both correlated with carcinoma infiltration of lymph nodes. MMP-13 also correlated with the expression of Her-2/neu (p = 0.015) and TIMP-1 (p < 0.010), respectively in tumor cells. Tumor-derived, but not stromal fibroblast-derived, MMP-13 correlated with aggressive tumor phenotypes. Moreover, high levels of MMP-13 expression were associated with decreased overall survival. In parallel, the prognostic value of MMP-13 expressed by peritumoral fibroblasts seems less significant. Our data suggest that lymph node status, tumor size, Her-2/neu expression, TIMP-1 and MMP-13 expression in cancer cells are independent prognostic factors.ConclusionTumor-derived, but not stromal fibroblast-derived, MMP-13 correlated with aggressive tumor phenotypes, and inversely correlated with the overall survival of breast cancer patients. MMP-13 may serve as an independent prognostic factor for invasive breast cancer patients. MMP-13 may be particularly useful as a prognostic marker when evaluated along with Her-2/neu and lymph node status.

Interaction between Cancer Cells and Stromal Fibroblasts Is Required for Activation of the uPAR-uPA-MMP-2 Cascade in Pancreatic Cancer Metastasis

Interaction between tumor cells and surrounding stromal fibroblast (SF) plays a critical role in tumor growth and invasion. The aim of the study is to determine the role of SF in regulating the invasive behaviors of pancreatic cancer by evaluating the mode of SF activating the urokinase plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP)-2 cascade. The expression patterns of uPA, MMP-2, and uPA receptor (uPAR) in human metastatic pancreatic cancer were analyzed by immunohistochemistry and the roles of SF in activation of the uPA-plasmin-MMP-2 cascade were evaluated by coculturing pancreatic cancer cell lines with SF. uPA expression and fibroblastic uPAR expression were correlated with liver metastasis of human pancreatic cancer. MMP-2 rather than MMP-9 was activated in the metastatic pancreatic cancer. In the in vitro culture system, the coculture of peritumor fibroblasts with metastatic pancreatic cancer BxPc3 cells resulted in activation of MMP-2 and up-regulation of uPAR expression. In this coculture system, the uPA-plasminogen cascade was involved in MMP-2 activation. This activation required a direct interaction between SF and cancer cells. In the coculture system, intergrin alpha(6)beta(1) expression was increased in BxPc3 cells, and blocking the function of integrin alpha(6)beta(1) decreased the activation of uPA and MMP-2. This suggests that interaction between integrins of cancer cells and the uPARs of the SF might be involved in the activation of the uPAR-uPA-MMP-2 cascade. Our results suggest that SF plays a role in promoting pancreatic cancer metastasis via activation of the uPA-plasminogen-MMP-2 cascade.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in pancreatic neoplasm and pancreatic stellate cells

EMMPRIN (extracellular matrix metalloproteinase inducer, CD147) participates in the progression of various malignancies by stimulating the synthesis of specific matrix metalloproteinases (MMP) from peritumoral fibroblasts. In the present study, the expression and functional role of EMMPRIN was investigated in pancreatic neoplasm. QRT-PCR, immunohistochemistry, immunoblot, and ELISA analyses were used to analyze the expression, localization, and release of EMMRPIN. Silencing of EMMPRIN was performed using siRNA oligonucleotides, and functional consequences were assessed using growth assays, invasion assays, as well as MMP1/MMP2 and VEGF ELISA. EMMPRIN mRNA levels were 2.2-fold increased in pancreatic cancer (n=52) and 2.0-3.5-fold increased in other pancreatic neoplasm (n=105), but unchanged in chronic pancreatitis (n=10) compared to normal pancreatic tissues (n=9). Strong and predominantly membranous immunostaining was observed in the cancer cells and surrounding stromal cells. EMMPRIN serum levels were also significantly increased in pancreatic cancer patients (n=44) (4.13 +/- 0.28 ng/ml) with an AUC of 0.97 compared to healthy volunteers (n=29) (0.95 +/- 0.16 ng/ml; p

Peritumoral Fibroblast SPARC Expression and Patient Outcome With Resectable Pancreatic Adenocarcinoma

SPARC (secreted protein acidic and rich in cysteine) is a protein involved in cell matrix interactions, wound repair, and cell migration, and has been reported to inhibit cancer growth. SPARC undergoes epigenetic silencing in many pancreatic cancers, but stromal fibroblasts adjacent to infiltrating pancreatic adenocarcinomas frequently express SPARC. We evaluated the prognostic significance of tumor and peritumoral SPARC expression in patients with pancreatic adenocarcinoma. The expression patterns of SPARC were characterized by immunohistochemistry in 299 primary pancreatic ductal adenocarcinoma resection specimens from patients who underwent pancreaticoduodenectomy at Johns Hopkins Hospital (Baltimore, MD) between 1998 and 2003. Kaplan-Meier analysis and Cox proportional hazards regression modeling were used to assess the mortality risk associated with the presence or absence of tumor SPARC and peritumoral SPARC status. By Kaplan-Meier analysis, patients whose pancreatic cancer stromal fibroblasts expressed SPARC (median survival, 15 months) had a significantly worse prognosis than patients whose tumor stroma did not express SPARC (median survival, 30 months; log-rank P < .001). In contrast, the expression of SPARC in pancreatic cancer cells was not associated with prognosis (log-rank P = .13). Controlling for other prognostic factors (tumor size, positive lymph nodes, margin status, tumor grade, and age), the relative hazard for patients whose stroma expressed SPARC compared with those whose stroma did not was 1.89 (95% CI, 1.31 to 2.74); the expression of SPARC in pancreatic cancer cells remained unrelated to prognosis (relative hazard, 1.02; 95% CI, 0.73 to 1.42). The expression of SPARC by peritumoral fibroblasts portends a poorer prognosis for patients with pancreatic cancer.

Emmprin in epithelioid sarcoma: Expression in tumor cell membrane and stimulation of MMP‐2 production in tumor‐associated fibroblasts

Emmprin is a transmembrane glycoprotein on tumor cells that stimulates peritumoral fibroblasts to produce matrix metalloproteinases (MMPs). Emmprin and the induced MMPs play a crucial role in tumor progression, invasion and metastasis of human carcinomas (epithelial malignancies). However, only a few reports have addressed its role in soft tissue sarcomas. This study investigated the expression and role of emmprin in epithelioid sarcoma (ES). Immunoblot studies of 2 ES cell lines showed that they express emmprin, and co-culture of these ES cells with dermal fibroblasts resulted in upregulation of gelatinase A (MMP-2) in fibroblasts, as shown by zymography, immunoblotting and enzyme immunoassay. This stimulation was inhibited by an activity-blocking peptide against emmprin and by antiemmprin antibody. In addition, in vivo, immunohistochemical analysis of 5 ES patient cases demonstrated diffuse emmprin expression in ES cells and MMP-2 expression in both ES cells and peritumoral fibroblasts. The histopathological findings that peritumoral fibroblasts that were not in direct contact with emmprin-expressing ES cells exhibit upregulated MMP-2 prompted us to look for a soluble form of emmprin. Soluble full-length emmprin released from ES cells was detected in conditioned medium and shown to stimulate MMP-2 production by fibroblasts. In conclusion, emmprin is expressed in ES in both membrane and soluble forms and stimulates MMP-2 production via interactions with fibroblasts, which could play a role in ES cell stromal invasion and vascular involvement.

High incidence of EMMPRIN expression in human tumors

Extracellular matrix metalloproteinase inducer expressed by tumor cells stimulates peritumoral fibroblasts to produce matrix metalloproteinases, thus contributing to tumor invasion and metastasis. To assess its suitability as potential therapeutic target, the overall incidence of EMMPRIN expression in normal and neoplastic tissues was analyzed. EMMPRIN expression was detected immunohistochemically using monoclonal antibodies MEM-M6/1 and HIM6 and tissue microarrays with 2,348 and 608 tissue samples from 129 distinct tumor types and 76 different normal tissues, respectively. Expression and glycosylation state of EMMPRIN in human breast cancer cells were analyzed by Western blot analysis with monoclonal antibodies recognizing distinct carbohydrate structures and biochemical methods. EMMPRIN expression was found in 112 of 129 tumor entities analyzed with malignant tumors being EMMPRIN positive more frequently than benign tumors. A remarkable heterogeneity in EMMPRIN expression between tumor entities was observed. Among others, squamous-cell carcinomas (60-100%), pancreatic (87%), chromophobic kidney (83%), hepatocellular (83%) or medullary breast (83%) adenocarcinomas as well as glioblastoma multiforme (79%) presented with a particular high incidence of EMMPRIN expression. There were a limited number of EMMPRIN-positive normal cell types including proliferatively active and differentiating epithelial cells, germ cells, myocardial cells in the left heart ventricle or vascular endothelial cells of the brain. We could further demonstrate that breast cancer cells expressed EMMPRIN isoforms differing in the presence or absence of Lewis X glycan structures. Our results may assist in defining the suitability of EMMPRIN as therapeutic target and predicting negative side effects.