In situ mRNA hybridization technique for analysis of metastasis-related genes in human colon carcinoma cells.

The purpose of this study was to determine whether the expression level of several genes that regulate different steps of the metastatic process correlates with the metastatic potential of human colon carcinoma cells. The mRNA expression level for epidermal growth factor receptor (growth), basic fibroblast growth factor and interleukin-8 (angiogenesis), type IV collagenase (invasion), E-cadherin and carcinoembryonic antigen (adhesion), and the multidrug resistance gene mdr-1 (drug resistance) in the human KM12 colon carcinoma cell lines and clones with different metastatic potential was measured by Northern blot analysis and by in situ hybridization technique. Highly metastatic KM12SM and KM1214 cells growing in culture uniformly expressed high levels of epidermal growth factor receptor, basic fibroblast growth factor, and carcinoembryonic antigen mRNA, whereas cultures of low metastatic KM12C, clone 1, clone 3, and clone 6 cells displayed heterogeneous patterns of expression. KM12C (low metastatic) and KM12SM (highly metastatic) cells were implanted into the subcutis (ectopic) or the wall of the cecum (orthotopic) of nude mice. The mRNA expression level for epidermal growth factor receptor, basic fibroblast growth factor, interleukin-8, type IV collagenase, carcinoembryonic antigen, and mdr-1 was increased in the cecal wall tumors as compared with subcutaneous tumors or in vitro cultures. These data demonstrate a direct correlation between constitutive and inducible expression of several metastasis-related genes and the metastatic potential of human colon carcinoma cells.     

Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma.

We examined the expression level of several genes that regulate different steps of metastasis in formalin-fixed, paraffin-embedded archival specimens of primary human colon carcinomas from patients with at least 5 years of follow-up. The expression of epidermal growth factor receptor, basic fibroblast growth factor, type IV collagenase, E-cadherin, and multidrug resistance (mdr-1) was examined by a colorimetric in situ mRNA hybridization technique concentrating on reactivity at the periphery of the neoplasms. The in situ hybridization technique revealed inter- and intratumor heterogeneity for expression of the metastasis-related genes. The expression of basic fibroblast growth factor, collagenase type IV, epidermal growth factor receptor, and mdr-1 mRNA was higher in Dukes's stage D than in Dukes' stage B tumors. Among the 22 Dukes' stage B neoplasms, 5 specimens exhibited a high expression level of epidermal growth factor receptor, basic fibroblast growth factor, and collagenase type IV. Clinical outcome data (5-year follow-up) revealed that all 5 patients with Dukes' stage B tumors developed distant metastasis (recurrent disease), whereas the other 17 patients with Dukes' stage B tumors expressing low levels of the metastasis-related genes were disease-free. Multivariate analysis identified high levels of expression of collagenase type IV and low levels of expression of E-cadherin as independent factors significantly associated with metastasis or recurrent disease. More specifically, metastatic or recurrent disease was associated with a high ratio (> 1.35) of expression of collagenase type IV to E-cadherin (specificity of 95%). Collectively, the data show that multiparametric in situ hybridization analysis for several metastasis-related genes may predict the metastatic potential, and hence the clinical outcome, of individual lymph-node-negative human colon cancers.     

Increased metastatic potential in human prostate carcinoma cells by overexpression of arachidonate 12-lipoxygenase

Arachidonate 12-lipoxygenase (LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid (HETE), a bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. Alteration in 12-LOX expression or activity has been reported in various carcinomas including prostate carcinoma. However, little is known about the impact of the altered expression or activity of 12-LOX on tumor metastasis. In the present study, we examined whether or not an increase in 12-LOX expression in human prostate carcinoma cells can modulate their metastatic potential. We report that increased expression of 12-LOX in PC-3 cells caused a significant change in cell adhesiveness, spreading, motility, and invasiveness. Specifically 12-LOX transfected PC-3 cells were more adhesive toward vitronectin, type I and IV collagen, but not to fibronectin or laminin, than cells transfected with control vector. Increased spreading on vitronectin, fibronectin, collagen type I and IV also was observed in 12-LOX transfected PC-3 cells when compared to control PC-3 cells. The increased spreading of 12-LOX transfected PC-3 cells was blocked by treatment with 12-LOX inhibitors, baicalein and CDC. 12-LOX transfected PC-3 cells were more invasive through Matrigel than cells transfected with control vector. In vivo, tumor cell invasion to surrounding muscle or fat tissues was more frequent in nude mice bearing s.c. tumors from 12-LOX transfected PC-3 cells than in those from control vector transfected cells. When injected via the tail vein into SCID mice with implanted human bone fragments, there was an increase in tumor metastasis to human bone by 12-LOX transfected PC-3 cells in comparison to control vector transfected cells. Taken together, our data suggest that an increase in 12-LOX expression enhances the metastatic potential of human prostate cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genes of laminin B1 chain, alpha 1 (IV) chain of type IV collagen, and 72-kd type IV collagenase are mainly expressed by the stromal cells of lung carcinomas.

In this study, we analyzed the expression of messenger (m)RNAs for laminin B1 chain, alpha 1 (IV) chain of type IV collagen, and 72-kd type IV collagenase in 15 primary lung carcinomas and in two metastatic adenocarcinomas to the lung. The results show that the mRNA synthesis for these proteins mainly occurs in the stromal fibroblasts and endothelial cells. In a proportion of tumors, mRNAs for laminin B1 chain and 72-kd type IV collagenase could also be observed in carcinoma cells, but the amount of mRNAs was considerably lower in them than in the stromal cells. There were no convincing signals for the presence of the alpha 1 (IV) chain of type IV collagen mRNA in any of the carcinoma cells. A simultaneous expression or lack of expression of signals for laminin B1 chain and 72-kd type IV collagenase mRNAs could be observed in carcinoma cells of 12 cases, suggesting that the activation of these two genes may be somehow connected. There was no association between the mRNA expression and the differentiation degree or the size of the tumors. The occurrence of the mRNA synthesis for the 72-kd type IV collagenase in stromal fibroblasts and endothelial cells indicates that the stromal cells of tumors have a more pronounced impact on the spread of the neoplastic disease than previously thought. The results further show that in their ability to synthesize these proteins the stromal cells of tumors resemble those of developing embryonic tissues. This resemblance is probably connected with the constant remodeling of extracellular matrix in response to the proliferative activity of carcinoma cells.     

Increased expression of the 72-kd type IV collagenase in prostatic adenocarcinoma. Demonstration by immunohistochemistry and in situ hybridization.

The expression of the 72-kd type IV collagenase has been implicated as an important factor in determining the invasive potential of malignant tumors. Using immunohistochemistry and nonisotopic in situ hybridization, type IV collagenase expression was assessed in benign and malignant prostatic tissue obtained from 117 surgical and autopsy specimens. Diffuse strong staining for type IV collagenase mRNA and protein was identified in the malignant cells of more than 85% of prostatic adenocarcinomas and the dysplastic cells of high grade prostatic intraepithelial neoplasia. Benign hyperplastic epithelium showed moderate expression in basal cells and mild expression in secretory cells. The qualitative patterns of type IV collagenase expression in prostatic epithelium at the protein and mRNA levels in individual cases were identical. There was no correlation between the level of type IV collagenase expression and either tumor grade or stage. In 10% of adenocarcinomas, focal mild to moderate stromal cell immunoreactivity was present but mRNA was not detectable in the stromal compartment in any case. The enhanced expression of type IV collagenase in dysplastic epithelium and prostatic adenocarcinoma suggests it contributes to the development of the invasive phenotype. The vast majority of the enzyme present in these tumors is synthesized by malignant cells and its production by stromal cells is negligible.     

Organ site-dependent expression of basic fibroblast growth factor in human renal cell carcinoma cells.

We investigated the influence of organ microenvironment on the angiogenic phenotype in human renal cell carcinoma (HRCC) cells. HRCC line SN12C was established in vitro from a surgical specimen, and metastatic line SN12PM6 was isolated from a lung metastasis produced by parental cells implanted into the kidney of nude mice. SN12C (low metastasis) and SN12PM6 (high metastasis) cells were injected into the kidney or subcutis of nude mice. The kidney tumors were highly vascularized (as revealed by immunohistochemistry using antibodies against factor VIII), and metastatic, whereas the subcutaneous tumors were not. The expression of mRNA for basic fibroblast growth factor (bFGF) in kidney tumors was 10 to 20 times that found in subcutaneous tumors. Similar data were obtained at the protein level by using fluorescence activated cell sorting, immunohistochemistry, and enzyme-linked immunosorbent assay. bFGF was detected in the urine of mice with tumors in the kidney but not subcutaneous tumors. The level of bFGF in the serum of mice with kidney tumors was two to three times that in mice with subcutaneous tumors. The changes in bFGF expression in the tumors was transient. Collectively, these data indicate that the organ microenvironment can influence the expression level of bFGF in HRCC.     

Requirement for matrix metalloproteinase-9 (gelatinase B) expression in metastasis by murine prostate carcinoma.

Although a number of effective therapies are available for localized prostate cancer, metastatic prostate cancer is difficult to treat and impossible to cure. Identification of the gene products that enable a prostatic carcinoma cell to metastasize should facilitate an understanding of the processes leading to metastasis. To characterize the contribution of matrix metalloproteinase-9 (MMP-9, gelatinase B or the 92-kd type IV gelatinase/collagenase) to the development of metastasis in prostate cancer, we reduced MMP-9 expression in metastatic murine prostatic carcinoma cells using a ribozyme. The ribozyme transfected cells had lower basal levels of MMP-9 as well as decreased levels after stimulation by transforming growth factor-beta or phorbol 12-myristate 13-acetate when compared with the parental cells or with control transfectants. The cells with down-regulated MMP-9 were unable to form lung colonies in the experimental metastasis assay, whereas the controls and parental cells readily formed metastases. All cell types readily formed tumors after injection and down-regulation of MMP-9 did not adversely affect the rate of tumor growth. Thus, MMP-9 expression is required for hematogenous metastasis in a murine prostate model system raising the possibility that it may play an equivalent role in human prostate cancer.     

Messenger RNA for two type IV collagenases is located in stromal cells in human colon cancer.

The gene expression of two type IV collagen-degrading enzymes (72-kd and 92-kd type IV collagenases) was investigated in human colon adenocarcinomas by in situ hybridization. In all cases (18 out of 18), messenger RNA for the 72-kd type IV collagenase was present and located in numerous fibroblasts in the stroma surrounding the invasive cancer tissue. In normal-appearing colonic mucosa distant from the cancer tissue, either no expression or only very weak expression of this enzyme was detected. Also the 92-kd type IV collagenase was found in all samples investigated (10 out of 10), exclusively expressed by tissue macrophages. A very strong hybridization signal for messenger RNA for the 92-kd enzyme was found in a subpopulation of tissue macrophages surrounding invading malignant epithelium. In normal-appearing colon tissue, a markedly weaker hybridization signal was observed in macrophages contained in Peyer's patches. No hybridization signals for either of the two type IV collagenases were detected in cancer cells. Together with previous findings on expression of components of the plasminogen activation system, these results indicate that several nonepithelial cell types in the tumor stroma are involved in production of factors involved in extracellular proteolysis during colon cancer invasion.     

应用RNA干扰技术研究激素非依赖性高转移潜能前列腺癌细胞中survivin的表达及意义

目的研究 survivin 在激素非依赖性高转移潜能前列腺癌中的表达及其与前列腺癌的生物学行为及侵袭和转移潜能的相关性。方法应用 RNA 干扰技术构建 survivin 真核表达载体,转染人激素非依赖性前列腺癌高转移亚系 PC-3M-1E8细胞系。通过细胞生长曲线、肿瘤细胞裸鼠异种接种、软琼脂集落形成实验检测体内、体外细胞生长能力;流式细胞术检测 survivin RNA 干扰质粒对细胞周期及细胞凋亡的影响,Western blot 检测 caspase3活性片段,观察 survivin 抑制细胞凋亡的情况;Matrigel 穿膜实验检测肿瘤细胞体外侵袭能力。结果稳定转染 survivin RNA 干扰质粒的 PC-3M-1E8细胞中 survivin 的 mRNA 及蛋白水平明显降低,蛋白水平与阴性对照组相比,约下降78%～80%,差异有统计学意义(P<0.01);体外培养细胞生长速度及裸鼠体内肿瘤生长速度均明显减慢,锚着不依赖性生长的能力(软琼脂克隆形成数:14.33±3.51)与阴性对照组(52.33±6.81)及空白对照组(54.00±6.00)相比明显降低(P<0.01);凋亡细胞比例明显增加,空白对照组、阴性对照组及干扰阳性组的凋亡比例分别为5.88±0.99、6.97±1.60、16.40±1.95,干扰阳性组的凋亡比例显著高于对照组(P<0.01),并伴有 caspase3活性片段的表达增加;细胞阻滞在 G_0/G_1期(干扰阳性组、阴性对照组及空白对照组的细胞 G_0/G_1期的比例分别为52.71±1.10、43.59±1.83及43.65±3.44,P<0.05),并在细胞形态学上出现多核巨细胞现象;细胞体外侵袭能力明显降低,干扰阳性组的细胞(38.67±6.59)与阴性对照组(46.07±9.97)及空白对照组(47.87±9.58)相比穿膜细胞数明显减少(P<0.05)。结论 survivin 在激素非依赖性高转移潜能前列腺癌中高表达,并与细胞凋亡、细胞生长及肿瘤侵袭有关。抑制 survivin 的表达可能成为临床治疗激素非依赖性前列腺癌的方法之一。     

Development of seven new human prostate tumor xenograft models and their histopathological characterization.

Seven human prostate tumor models were established by transplanting tumor fragments in NMRI athymic nude mice. Once established, the tumors were serially transplantable in both NMRI and BALB/c nude mice. The xenografts originated from primary prostatic carcinomas (prostatectomy specimens), transurethral resection material, and metastatic lesions (pelvic lymph nodes and scrotal skin). Histological examination revealed that, in the course of several mouse passages (8 to 23), tumors retained their resemblance to the original patient material. The PC-295, PC-310, PC-329, and PC-346 tumors are dependent on androgens for their growth. The PC-324, PC-339, and PC-374 tumors are androgen independent, although growth of PC-374 tumors still seemed androgen sensitive. All tumors are diploid, except for the PC-374, which is tetraploid. The diploid PC-295 tumor has an additional small population of tetraploid cells. All xenografts displayed a heterogeneous expression pattern of the androgen receptor except for the PC-324 and PC-339 tumors in which the androgen receptor could not be detected. Prostatic acid phosphatase and prostate-specific antigen were retained during serial transplantation in all tumors but the PC-324 and PC-339. This panel of permanent human prostate tumor models comprises tumors representing both the androgen-dependent and -independent stages of human prostate cancer with various degrees of differentiation and, therefore, is of great value for the study of many aspects of growth and progression of human prostate cancer.     

Basic fibroblast growth factor regulates type I collagen and collagenase gene expression in human smooth muscle cells.

Basic fibroblast growth factor (bFGF) is a multifunctional peptide well known for angiogenic, neurotropic, and mesoderm-inducing effects. In the present study, we have investigated the effects of bFGF on collagen and collagenase gene expression in human iliac arterial smooth muscle cells. We report that bFGF inhibits type I collagen gene expression and collagen biosynthesis, with concomitant stimulation of collagenase gene expression. The smooth muscle cells incubated with human recombinant bFGF decreased the mRNA steady state levels of pro-alpha 1(I) type I collagen by as much as 72%. [3H]Hydroxyproline synthesis was also suppressed by 59% compared with untreated control cultures. Indirect immunofluorescence confirmed corresponding changes at the protein level. In contrast to the down-regulation of type I collagen gene expression, collagenase gene expression was found to be up-regulated severalfold by bFGF. The data suggest that bFGF is capable of regulating collagen and collagenase gene expression divergently in human smooth muscle cells and that the effects appear to be mediated at a pretranslational level.     

LNMAT1/CADM1调控轴对前列腺癌侵袭和免疫抑制因子的影响

目的研究非编码RNA淋巴结转移相关转录本1/细胞黏附分子1(LNMAT1/CADM1)调控轴对前列腺癌(prostate adenocarcinoma,PRAD)细胞侵袭和免疫抑制因子表达水平的影响。方法收集临床PRAD和癌旁(TAC)的组织。构建LNMAT1的慢病毒短发夹RNA(shRNA)载体和阴性对照(NC-shRNA)、CADM1 siRNA(si-CADM1)和阴性对照(NC-siRNA)并转染人高转移性前列腺癌细胞PC-3M。PC-3M细胞处理分组为:NC-shRNA组、LNMAT1 shRNA组、LNMAT1 shRNA+si-CADM1组、LNMAT1 shRNA+NC-siRNA组。实时荧光定量PCR(RT-qPCR)检测PRAD组织、TAC组织、人前列腺上皮细胞RWPE-1、人前列腺癌细胞DU145、PC-3M中的LNMAT1和CADM1的表达。RT-qPCR检测PC-3M分组中LNMAT1、CADM1、MMP-2,MMP-9,E-cadherin和N-cadherin的水平。蛋白免疫印迹法检测PC-3M分组中CADM1、TGF-β、IL^-10、VEGF-A、FasL、HLA-G的水平。伤口愈合检测PC-3M细胞的迁移率,Transwell法检测细胞侵袭率。结果 LNMAT1在PC-3M细胞和有淋巴转移的PRAD组织中上调(均为P<0.05);沉默LNMAT1不仅抑制PC-3M细胞侵袭和迁移能力(均为P<0.05),下调MMP-2、MMP-9、N-cadherin的mRNA水平(均为P<0.05),而且上调E-cadherin的mRNA(P<0.05)。另外,沉默LNMAT1抑制PC-3M细胞免疫抑制因子TGF-β、IL^-10、VEGF-A、FasL、HLA-G的蛋白水平(均为P<0.05);LNMAT1负调控PC-3M细胞中CADM1的表达(P<0.05);沉默CADM1显著逆转PC-3M细胞中LNMAT1对侵袭和免疫抑制因子的抑制能力(均为P<0.05)。结论本研究阐明LNMAT1/CADM1调控轴可影响高转移性PRAD细胞的侵袭和免疫抑制因子。     

Tumour-stroma interactions between metastatic prostate cancer cells and fibroblasts

Previous work has shown the importance of tumour-stroma interactions for prostate cancer development at the primary site. The aim of the present study was to find out whether evidence can be found for a tumour-stroma cross- talk also between metastatic prostate cancer cell lines and non-prostatic stromal fibroblasts which are encountered by metastatic cells at most sites. We addressed this issue in cell culture systems using 3 metastatic human prostate cancer cell lines (LnCaP, PC-3 and DU-145) on the one hand, and a human fibroblast line (HFF, human foreskin fibroblasts) on the other. We incubated fibroblasts with tumour cell- and tumour cells with fibroblast-conditioned media and evaluated several parameters important for the establishment of metastases such as cell proliferation, migration and expression of matrix degrading proteases. We also determined in the conditioned media the concentrations of several growth factors and cytokines which might be responsible for the observed effects. We found that media conditioned by all 3 metastatic prostate cancer cell lines stimulated fibroblast proliferation which corresponds to fibrous stroma induction in vivo. DU-145 cell conditioned media induced in fibroblasts expression of mmp-1 mRNA known to be important for tumour invasion. ELISA assays revealed that tumour cells secrete bFGF, PDGF and TNFalpha known to stimulate fibroblast proliferation and/or MMP-1 expression. Cultivation of DU-145 carcinoma cells in fibroblast conditioned medium resulted in an enhanced proliferation and anchorage-independent growth of this cell line in soft agar. Fibroblast conditioned medium also increased migration of PC-3 cells in the wound assay and slightly augmented mmp-1 expression. KGF (able to stimulate proliferation of normal and neoplastic prostate epithelial cells) was secreted by fibroblasts at higher concentrations than by all 3 tumour cell lines. In addition, fibroblasts secreted TNFalpha, bFGF, PDGF, HGF and also VEGF, the most important factor for tumour vascularization. Our results provide evidence that tumour-stroma interactions do not only exist at the primary site but also between metastatic prostate cancer cell lines and their fibroblastic microenvironment. These interactions, which are mediated through secreted factors, affect several steps of the metastatic cascade including proliferation, anchorage-independent growth, migration and the secretion of matrix-degrading proteases.     

In vivo monitoring of tumor relapse and metastasis using bioluminescent PC-3M-luc-C6 cells in murine models of human prostate cancer

We used the bioluminescent human prostate carcinoma cell line PC-3M-luc-C6 to non-invasively monitor in vivo growth and response of tumors and metastasis before, during and after treatments. Our goal was to determine the utility of a luciferase-based prostate cancer animal model to specifically assess tumor and metastatic recurrence in vivo following chemotherapy. Bioluminescent PC-3M-luc-C6 cells, constitutively expressing luciferase, were implanted into the prostate or under the skin of mice for primary tumor assessment. Cells were also injected into the left ventricle of the heart as an experimental metastasis model. Weekly serial in vivo images were taken of anesthetized mice that were untreated or treated with 5-fluorouracil or mitomycin C. Ex vivo imaging and/or histology was used to confirm and localize metastatic lesions in various tissues initially detected by images in vivo. Our in vivo data detected and quantified early inhibition of subcutaneous and orthotopic prostate tumors in mice as well as significant tumor regrowth post-treatment. Local and distal metastasis was observed within seven days following intracardiac injection of PC-3M-luc-C6 cells. Differential drug responses and metastatic tumor relapse patterns were distinguished over time by in vivo imaging depending on the metastatic site. The longitudinal evaluation of bioluminescent tumor and metastatic development within the same cohorts of animals permitted sensitive and quantitative assessment of both primary and metastatic prostate tumor response and recurrence in vivo.