Matrix metalloproteinase-7 triggers the matricrine action of insulin-like growth factor-II via proteinase activity on insulin-like growth factor binding protein 2 in the extracellular matrix.

Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM-bound growth factor or cytokine activation have not been well documented. We showed that the insulin-like growth factor type-1 receptor (IGF-1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)-7 to a serum-starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF-II neutralizing antibody. In the ECM of this cell line, IGF-II and IGF binding protein (BP)-2 coexisted, but IGFBP-2 disappeared from the ECM fraction after treatment with MMP-7 or heparinase III. On the other hand, in a cell line in which IGF-1R was overexpressed, IGF-1R was phosphorylated by supernatant from the MMP-7-treated ECM fraction of HT29 but not by that from a heparinase-III-treated ECM fraction. We also demonstrated that MMP-7 degrades IGFBP-2 in vitro at three cleavage sites (peptide bonds E(151)-L(152), G(175)-L(176) and K(181)-L(182)), which have not been documented previously. Taken together, these results demonstrate that MMP-7 generates bioactive IGF-II by degrading the IGF-II/IGFBP-2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF-II-induced signal transduction. This evidence indicates that some ECM-associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action ('protease-triggered matricrine') represents an attractive model for understanding ECM-tumor interactions.

Expression of tumor suppressor and tumor-related proteins in differentiated carcinoma, undifferentiated carcinoma with tubular component and pure undifferentiated carcinoma of the stomach.

BACKGROUND: The recent development of tissue microarray (TMA) technology allows high-throughput protein expression profiling of cancer tissues by immunohistochemistry. We attempted to clarify the derivation of undifferentiated-type gastric carcinoma with tubular component by using TMA. METHODS: We constructed a TMA system composed of six paraffin blocks in which 274 samples of formalin-fixed gastric carcinoma tissue from 274 patients were embedded. Using this system, we performed immunohistochemical stains for five tumor suppressor and tumor-related proteins, i.e. p53, p16, hMLH1, c-erbB-2 and carcinoembryonic antigen (CEA). The 274 gastric carcinomas were histopathologically divided into the following three groups according to the degree of differentiation: differentiated-type (D-type), undifferentiated-type with tubular component (UT-type) and pure undifferentiated-type (UP-type). Immunohistochemical results were then compared with histological types. RESULTS: The percentages of abnormal expression of each protein in D-type, UT-type and UP-type carcinomas were as follows: 27% (38/143), 17% (17/98) and 15% (5/33) for p53; 27% (39/143), 19% (19/98) and 18% (6/33) for p16; 38% (54/143), 44% (43/98) and 24% (8/33) for hMLH1; 15% (22/143), 5% (5/98) and 0% (0/33) for c-erbB-2; and 22% (31/143), 35% (34/98) and 70% (23/33) for CEA. UP-type carcinomas exhibited the lowest frequencies of abnormal expression for p53, p16, hMLH1 and c-erbB-2, but the highest frequencies for CEA. UT-type carcinomas generally showed intermediate frequencies between those of D-type and UP-type carcinomas. Differences between D-type and UP-type for c-erbB-2 (P < 0.05) and CEA (P < 0.001) were significant, as were differences between D-type and UT-type for c-erbB-2 (P < 0.05) and CEA (P < 0.05), and differences between UT-type and UP-type for hMLH1 (P < 0.05) and CEA (P < 0.001). CONCLUSIONS: These findings reveal that gastric carcinomas have distinct expression profiles for tumor suppressor and tumor-related proteins depending on histological types, and support the hypothesis that UT-type carcinomas are derived not only from D-type but also from UP-type carcinomas. We also found significant differences between abnormal protein expression and other clinicopathological parameters such as gender, age and status of tumor and nodes.

Histological characteristics of tumor cells and stromal cells in vessels and lymph nodes are important prognostic parameters of extrahepatic bile duct carcinoma: a prospective study.

Extrahepatic bile duct carcinomas (EHBDCs) consist of primary tumors, tumors in vessels, and tumors in lymph nodes. The purpose of this study was to prospectively investigate whether the histological characteristics of tumor cells and tumor stromal cells in vessels and lymph nodes were significantly associated with the outcomes of 60 EHBDC patients as compared with the histological characteristics of tumor cells and tumor stromal cells in primary tumors. Multivariate analyses, using the Cox proportional hazard regression model, showed that in EHBDCs without nodal metastasis, blood vessel tumor emboli with an angiomatous stroma significantly increased the hazard ratios (HRs) of tumor recurrence and death ( P < .05). In EHBDCs with nodal metastasis, the presence of tumor necrosis in the nodal tumors significantly increased the HRs of tumor recurrence and initial distant organ metastasis ( P < .05). In EHBDCs located in the distal to middle portion of the extrahepatic bile duct, blood vessel tumor emboli with an angiomatous stroma significantly increased the HRs of tumor recurrence, initial distant organ metastasis, and death ( P < .05). Severe nuclear atypia of the tumor cells in lymph vessels significantly increased the HRs of tumor recurrence and initial distant organ metastasis ( P < .05). In EHBDCs located in the hilar portion of the extrahepatic bile duct, the presence of nodal tumors with more than 4 mitotic figures significantly increased the HRs of tumor recurrence and initial distant organ metastasis ( P < .05). Several histological characteristics of tumor cells and tumor stromal cells in vessels and lymph nodes have significant effects on tumor progression of EHBDCs.

In vivo and in vitro characterization of human fibroblasts recruited selectively into human cancer stroma.

Fibroblasts, which are a major component of cancer-induced stroma, can have a significant impact on the progression of adjacent malignant epithelia. To characterize fibroblasts recruited into cancer-induced stroma, we examined the recruitment efficiency of 9 human fibroblast cell lines into experimental tumors generated in immunodeficient mice. Green fluorescence protein (GFP)-labeled fibroblast cell lines and human pancreatic cancer cell line Capan-1 were injected i.p. at different sites; the GFP-labeled cells within xenografts were then analyzed. KM104GFP (bone marrow) and VA-13GFP (lung) were selectively recruited into cancer stroma more efficiently than the other cell lines. KM104GFP cells did not affect tumor volume; however, VA-13GFP cells increased tumor volume by about 2-fold. After 5 cyclic in vivo passages of KM104GFP in Capan-1, we selected a subpopulation with an 8.4-fold higher recruitment efficiency (KM104GFP-5G) compared to parental KM104GFP. KM104GFP-5G also exhibited higher chemotaxis and chemoinvasion activity compared to KM104GFP in response to cancer-released chemoattractant(s). Oligonucleotide microarray analysis identified 8 genes with >3-fold upregulation and 6 genes with >3-fold downregulation in KM104GFP-5G. Immunohistochemistry confirmed that fibroblasts recruited into pancreatic cancer stroma strongly expressed carbonic anhydrase IX and keratin-8, whose transcripts were upregulated in KM104GFP-5G by oligonucleotide microarray analysis, whereas their expression in fibroblasts within noncancerous pancreatic stroma were under the detection level. Our results indicate that fibroblast recruitment is not selective with respect to organ origin and that particular fibroblast subpopulations with specific phenotypic characteristics could be recruited efficiently into cancer-induced stroma.

In vivo characterization of bone marrow-derived fibroblasts recruited into fibrotic lesions.

Fibroblasts, which are widely distributed and play a key part in tissue fibrosis, are phenotypically and functionally heterogeneous. Recent studies reported that bone marrow can be a source of tissue fibroblast. In the study reported here, we investigated in vivo characterization of bone marrow-derived fibroblasts recruited into various fibrotic lesions. Mice were engrafted with bone marrow isolated from transgenic mice expressing green fluorescent protein (GFP), and fibrotic lesions were induced by cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung). A small population of GFP+ fibroblast was found even in nonfibrotic skin (8.7% +/- 4.6%) and lung (8.9% +/- 2.5%). The proportion of GFP+ fibroblasts was significantly increased after cancer implantation(59.7% +/- 16.3%) and excisional wounding (32.2% +/- 4.8%), whereas it was not elevated after bleomycin administration (7.1% +/- 2.4%). Almost all GFP+ fibroblasts in fibrotic lesions expressed type I collagen, suggesting that bone marrow-derived fibroblasts would contribute to tissue fibrosis. GFP+ fibroblasts expressed CD45, Thy-1, and alpha-smooth muscle actin at various proportions. Our results suggested that bone marrow-derived fibroblasts expressed several fibroblastic markers in vivo and could be efficiently recruited into fibrotic lesions in response to injurious stimuli; however, the degree of recruitment frequency might depend on the tissue microenvironment.

Primary lung adenocarcinoma with massive lymphocyte infiltration.

We report 6 cases of adenocarcinoma with massive lymphocyte infiltration. This adenocarcinoma is found in 0.7% of surgically resected primary lung adenocarcinomas. The tumor was located in the peripheral portion in all cases. Lymph node metastasis was detected in 4 cases. The mean follow-up period was 49.8 months; all patients were alive without recurrence despite advanced pathologic stage. Histologically, the tumors revealed an acinar and glandular structure, and the cancer epithelium was destroyed by infiltrating lymphocytes. The lung parenchyma was destroyed by the severe inflammatory cell infiltration without dense fibrosis. Immunohistochemical staining revealed that most infiltrating lymphocytes were positive for CD3 or CD20. None of the tumors were positive for latent membrane protein-1, bcl-2, or Epstein-Barr virus-encoded small RNA-1. This type of adenocarcinoma occurs in old age, and good outcome and distinctive histologic features were observed. We refer to it as primary lung adenocarcinoma with massive lymphocyte infiltration.

Low levels of insulin-like growth factor type 1 receptor expression at cancer cell membrane predict liver metastasis in Dukes' C human colorectal cancers.

PURPOSE: The aim of this study was to evaluate the prognostic significance of insulin-like growth factor type 1 receptor (IGF-1R) expression in Dukes' C human colorectal cancers (CRCs). EXPERIMENTAL DESIGN: Immunohistochemical staining for IGF-1R was done on formalin-fixed, paraffin-embedded specimens from 161 patients with curatively resected Dukes' C CRC and at least 5-year follow-up periods. We investigated the association between the levels of IGF-1R expression and the clinicopathologic parameters. To evaluate the accurate prognostic value of IGF-1R expression, we investigated two patterns of recurrence-free survival (RFS) according to the mode of recurrence, the hepatic-RFS (H-RFS), and the nonhepatic-RFS (nH-RFS). The influence of the pattern of IGF-1R immunostaining (membranous or cytoplasmic) on RFS was also estimated. RESULTS: High (diffuse staining) and low (focal staining) levels of IGF-1R expression were found in 45 (28%) and 116 (72%) specimens, respectively. The recurrence rate was significantly higher in the latter group (49 of 116) than the former group (9 of 45; P = 0.01). H-RFS was significantly longer for the former group than the latter group (P = 0.021), whereas no difference was found in nH-RFS between the two groups (P = 0.121). In multivariate analysis, the level of IGF-1R expression was an independent factor for H-RFS (P = 0.015) as were the depth of invasion and lymph vessel invasion (P = 0.006 and 0.022, respectively). Using a combination of the level of IGF-1R expression and these two factors, the prognostic value was further increased. When IGF-1R staining patterns (membranous or cytoplasmic) were compared, membrane staining of IGF-1R possessed prognostic significance. CONCLUSIONS: In Dukes' C CRC, focal membrane expression of IGF-1R in the primary tumor can predict a high risk of recurrence, especially liver metastasis. Understanding the mechanisms involved could lead to new therapeutic approaches for advanced CRC.

Growth inhibition of human prostate cancer cells in human adult bone implanted into nonobese diabetic/severe combined immunodeficient mice by a ligand-specific antibody to human insulin-like growth factors.

Advanced prostate cancer frequently involves the bone that has the largest content of insulin-like growth factors (IGFs). However, the role of bone-derived IGFs in bone metastasis of prostate cancer has not been studied extensively because of the lack of a reliable animal model. Therefore, we investigated whether a novel antibody directed against human IGF-I and IGF-II (KM1468) could inhibit the development of new bone tumors and the progression of established bone tumors in nonobese diabetic/severe combined immunodeficient mice implanted with human adult bone. We first confirmed that KM1468 bound specifically to human IGF-I, human IGF-II, and mouse IGF-II but not to insulin. It also blocked autophosphorylation of the type I IGF receptor induced by the binding of IGFs in human-type I IGF receptor-overexpressing BALB/c 3T3 cells, and it inhibited the IGF-stimulated growth of MDA PCa 2b cells in vitro. Then mice were injected intraperitoneally with KM1468 once weekly for 4 weeks either immediately or 4 weeks after inoculation of MDA PCa 2b cells. KM1468 markedly and dose-dependently suppressed the development of new bone tumors and the progression of established tumor foci, as determined by histomorphometry, and it also decreased serum prostate-specific antigen levels, compared with the control. This is the first report of an IGF ligand-specific inhibitory antibody that suppresses the growth of human prostate cancer cells in human adult bone. These results indicate that the IGF signaling axis is a potential target for prevention and treatment of bone metastases arising from prostate cancer.

Nuclear beta-catenin accumulation as a prognostic factor in Dukes' D human colorectal cancers.

Beta-catenin is well recognized to play a crucial role as a transcriptional factor during the early step of colorectal carcinogenesis. Some reports concerning the clinical implications of cytoplasmic and/or nuclear beta-catenin accumulation are available, though their results vary. On the other hand, the clinical implication of nuclear accumulation of beta-catenin in Dukes' D colorectal cancers (with distant metastasis) has not been investigated. To assess its value as a prognostic marker in this stage, we selected the cases with synchronous liver metastasis. Thirty-eight surgically resected primary and corresponding metastatic liver tumors were examined immunohistochemically and the relationships between nuclear beta-catenin accumulation and clinicopathological variables were analyzed. Of the 38 primary colorectal cancers analyzed, 11 (29%) showed nuclear accumulation of beta-catenin with cytoplasmic staining. Nuclear accumulation positivity was more frequently associated with lymph node metastasis than being negative [100% (11/11) vs. 67% (18/27), p=0.04]. There was a significant difference in median survival time between the nuclear beta-catenin positive group (1130 days) and the negative group (2102 days: p=0.037). Interestingly, all of the patients (9/9) in the former group had died when the recurrence was in the liver, while 42% (8/19) in the latter group had survived even if the recurrence was in the liver (p=0.03). In conclusion, though these results were obtained in a small series of patients, nuclear accumulation of beta-catenin may be a useful prognostic marker even in Dukes' D colorectal cancers.

Primary lung carcinoma with signet-ring cell carcinoma components: clinicopathological analysis of 39 cases.

The clinical and histologic profiles of primary lung carcinomas with signet-ring cell carcinoma (SRCC) components were analyzed. The SRCC components were seen in 39 cases (1.5%) of 2640 cases of surgically resected primary lung carcinomas. The patients' mean age was 54.6 years (range, 32-76 years), and the male-to-female ratio was 1.16:1.00. Twenty-six patients (66.7%) were smokers. The SRCC component was seen as part of an adenocarcinoma in 36 cases (acinar, 27 cases; mixed bronchioloalveolar and acinar, 9 cases) and as part of an adenosquamous cell carcinoma in 2 cases; one lesion consisted of only SRCC cells. The morphologic appearance of cancer nests containing SRCC was classified into three patterns: solid, tubuloacinar, and discohesive. Each case was composed of a varying proportion of these three patterns. The carcinomas with SRCC components were divided into two groups, according to those in which the SRCC component occupied <50% of the lesion (L-SRCC; n = 20) and those in which the SRCC component occupied > or = 50% of the lesion (H-SRCC; n = 19). The frequencies of blood vessel invasion, lymph vessel invasion, and lymph node metastasis were significantly higher in the H-SRCC group than in 1634 adenocarcinoma, and adenosquamous cell carcinomas (non-SRCC). The 5-year survival rates of patients non-SRCC, with l-SRCC, or with H-SRCC were 52.7%, 50%, and 28.4%, respectively. The 5-year survival rate of patients with H-SRCC and of patients without SRCC were significantly different. Based on these clinicopathologic characteristics, we classified primary lung carcinomas with SRCC components into the following two groups: 1) SRCC, in which the SRCC component occupies >or = 50% of the lesion, and 2) signet-ring feature, in which the SRCC components occupies <50% of the lesion.

Matrix metalloproteinase-7 facilitates insulin-like growth factor bioavailability through its proteinase activity on insulin-like growth factor binding protein 3.

Matrix metalloproteinase-7 (MMP-7) secreted by cancer cells has been implicated classically in the basement membrane destruction associated with tumor cell invasion and metastasis. Recent epidemiologic studies have established a correlation between high levels of circulating insulin-like growth factor (IGF) and low levels of IGF binding protein 3 (IGFBP-3), and relative risk of developing colon, breast, prostate, and lung cancer, which are known to produce MMP-7. In this study, IGFBP-3 was assessed as a candidate for the physiologic substrate of MMP-7. MMP-7 proteolysis generated four major fragments (26 kDa, 17 kDa, 15.5 kDa, and 15.5 kDa), and two cleavage sites were identified: one at the site of hydrolysis of the K(144)-I(145) peptide bond and one at the R(95)-L(96) peptide bond. The former site is different from the previously reported site of cleavage of IGFBP-3 by other proteases. Addition of IGFBP-3 inhibited IGF-I-mediated IGF type 1 receptor (IGF-IR) phosphorylation and activation of the downstream molecule Akt in BALB/c 3T3 fibroblasts overexpressing human IGF-IR (3T3-IGF-IR) and in two human colon cancer cell lines (COLO201 and HT29). Coincubation of the IGF-I/IGFBP-3 complex with MMP-7 restored IGF-I-mediated IGF-IR phosphorylation and activation of Akt in these cell lines. The IGF-I signal recovered by MMP-7 protected against apoptosis induced by anoikis in 3T3-IGF-IR cells. These results indicate that MMP-7 proteolysis of IGFBP-3 plays a crucial role in regulating IGF-I bioavailability, thereby promoting cell survival. This mechanism may contribute to the tumorigenesis of MMP-7-producing IGF-IR-expressing tumors in the primary site and to organ-specific metastasis in a paracrine manner.

Predictive markers for late cervical metastasis in stage I and II invasive squamous cell carcinoma of the oral tongue.

PURPOSE: Patients with oral tongue carcinoma treated by intraoral excision only should be followed up carefully for cervical lymph node metastasis and salvaged immediately if found, because some patients have a more aggressive clinical course. The purpose of this study was to find useful markers for predicting late cervical metastasis in patients with stage I and II invasive squamous cell carcinoma of the oral tongue. EXPERIMENTAL DESIGN: We investigated clinicopathologic factors and immunohistochemical biomarkers predicting late cervical metastasis in surgical specimens from 56 patients with T(1-2)N(0)M(0) invasive squamous cell carcinoma of the oral tongue who did not undergo elective neck dissection. Histopathologic factors including tumor thickness, mode of invasion, Broders grade, total score of three different malignancy grading systems, eight other clinicopathologic parameters, and immunohistochemical expression of p53, cyclin D1, Ki-67, epidermal growth factor receptor, microvessel density, cyclooxygenase-2, MUC1, laminin-5 gamma2, E-cadherin, and beta-catenin were examined. All of the clinicopathologic factors and immunohistochemical expression of biomarkers were compared in terms of survival. RESULTS: In the univariate analysis, tumor thickness (P = 0.009), Broders grade (P = 0.017), nest shape (P = 0.005), mode of invasion (P < 0.001), Anneroth score (P = 0.029), Bryne score (P < 0.001), and E-cadherin expression (P = 0.003) were correlated with late cervical metastasis. Multivariate analysis on late cervical metastasis revealed that tumor thickness >4 mm, mode of invasion grade 3 or 4, and E-cadherin expression were independent factors. Late cervical metastasis was the only prognostic factor for overall survival (P = 0.002). CONCLUSIONS: Our results indicate that patients with stage I and II invasive squamous cell carcinoma of the oral tongue with tumor thickness >4 mm, mode of invasion grade 3 or 4, and low expression of E-cadherin should be considered a high-risk group for late cervical metastasis when a wait-and-see policy for the neck is adopted.

Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction.

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.

Histogenesis of hepatoid adenocarcinoma of the stomach: molecular evidence of identical origin with coexistent tubular adenocarcinoma.

Hepatoid adenocarcinoma of the stomach is a highly malignant neoplasm. Most gastric hepatoid adenocarcinomas coexist with tubular adenocarcinoma. However, the relationship between hepatoid adenocarcinoma and tubular adenocarcinoma is still unclear. In the present study, the characteristics of the coexistent tubular adenocarcinomas were determined by examining their profiles of mucin production. Subsequently, molecular pathological techniques were applied to determine the clonality of 15 mixed hepatoid and tubular adenocarcinomas of the stomach. Mucin analysis suggested that the coexistent tubular adenocarcinomas with hepatoid adenocarcinoma were of the intestinal type. The patterns of chromosome X inactivation were identical between the hepatoid adenocarcinoma component and tubular adenocarcinoma component in all of 3 informative female cases. Mutations in the p53 gene were found in 5 cases. The sequences were identical within both tumor components in all 5 cases. Microsatellite analysis indicated more than 3 common patterns of loss of heterozygosity in 8 cases. These observations strongly suggested that hepatoid adenocarcinomas were of identical origin to coexistent tubular adenocarcinomas.

Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer.

The PTEN tumor suppressor gene on 10q23.3, responsible for the Cowden and Bannayan-Zonana syndromes, encodes a dual-specificity phosphatase able to dephosphorylate both tyrosine phosphate and serine/threonine phosphate residues. Mutational inactivation of PTEN has been reported in various malignancies, including endometrial cancers, ovarian cancers, and glioblastomas. In this study, we investigated PTEN gene mutations in 10 gastric cancer cell lines and 58 primary gastric cancers by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP). Hypermethylation of promoter region CpG islands, an alternative mechanism of gene inactivation to coding region mutations, was also evaluated by methylation specific PCR (MSP). Only one (1.7%) of the 58 primary tumors carried a somatic 5-bp deletion in intron 7 of PTEN, which did not alter the mRNA sequence, and no mutations were detected in any of the cell lines. Similar levels of PTEN mRNA expression were observed in all cell lines and primary tumors studied by RT-PCR, and PTEN promoter CpG islands remained unmethylated. Therefore, we conclude that PTEN does not participate in gastric carcinogenesis as a tumor suppressor gene.