Acceleration of human prostate cancer growth in vivo by factors produced by prostate and bone fibroblasts.

Prostate cancer, the most prevalent cancer affecting men, frequently metastasizes to the axial skeleton where it produces osteoblastic lesions with growth rates often exceeding that of the primary tumor. To evaluate the role of tumor cell-host stromal interaction and stromal specific growth factors (GFs) in prostate cancer growth and progression, we coinoculated athymic mice with human prostate cancer cells (LNCaP) and various nontumorigenic fibroblasts s.c. LNCaP tumor formation was most consistently induced by human bone (MS) fibroblasts (62%), followed by embryonic rat urogenital sinus mesenchymal (rUGM) cells (31%) and Noble rat prostatic fibroblasts (17%), but not by NIH-3T3, normal rat kidney, or human lung CCD16 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. The human prostate component of these tumors was confirmed with immunohistochemical staining for prostate-specific antigen (PSA), Northern analysis for PSA expression, and Southern analysis for human repetitive Alu sequences. Elevations in serum PSA paralleled the histomorphological and biochemical findings. LNCaP and fibroblast cell-conditioned media (CM) was used to determine whether autocrine and paracrine mitogenic pathways exist between LNCaP and fibroblast cells in vitro, and various defined GFs were tested to identify possible active factors. Mitogenic assays revealed a 200-300% bidirectional stimulation between LNCaP and bone or prostate fibroblast-derived CM. Lung, normal rat kidney, and 3T3 fibroblast CM were not mitogenic for LNCaP cells. Among the purified GFs tested basic fibroblast growth factor (bFGF) was the most potent mitogen, stimulating LNCaP growth 180% in a concentration-dependent manner. Transforming growth factor alpha and epidermal growth factor were both minimally mitogenic. Coinoculation of LNCaP cells with a slowly absorbed matrix (Gelfoam) absorbed with bFGF or dialyzed and concentrated rUGM or MS CM was also capable of inducing LNCaP tumor formation in vivo. These observations illustrate that fibroblasts differentially modulate prostate cancer growth through the release of paracrine-mediated GFs, possibly including bFGF, and that tumor-stromal cell interactions play an important role in prostate cancer growth and progression.     

Naturally produced extracellular matrix inhibits growth rate and invasiveness of human osteosarcoma cancer cells

Enhanced supplementation with certain naturally occurring nutrients and vitamins has been associated with a reduction in occurrence and progression of human cancer. The exact mechanisms of this action are still under investigation. Extracellular matrix (ECM) plays a key role in the development of cancer. Therefore, we studied whether nutrients could exert anticancer effects through alteration of ECM biological properties. Confluent cultures of normal human dermal fibroblasts were allowed to produce and deposit ECM during a 7 d incubation period in the presence of tested compounds. Subsequently, fibroblasts were removed and the growth rates of freshly placed cultures of human osteosarcoma cells (lines U2-OS, MMNG, or SK.ES1) on pre-formed ECM were assayed in plain cell growth medium. In addition, ECM was deposited by fibroblasts on the upper surface of a porous plastic membrane and the subsequent migration of osteosarcoma cells to the other side was assayed in non-supplemented medium. The results demonstrated that the ECM produced by normal fibroblasts treated with a mixture of ascorbic acid, lysine, proline, arginine, cysteine, and green tea polyphenols significantly reduced the growth rate and invasive activity of osteosarcoma cells in contrast to the non-supplemented control. The changes in ECM properties were accompanied by significant changes in ECM protein and glycosaminoglycan composition as assayed immunochemically. We conclude that anticancer effects of nutrients involve beneficial changes in ECM biological properties. The role of ECM components in ECM-dependent regulation of tumor cell activities is discussed.     

Down-regulation of the extracellular matrix protein SPARC in vSrc- and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo

In vitro transformation of primary cultures of chick embryo fibroblasts by the membrane-bound vSrc or the nuclear vJun oncoproteins is correlated with a down-regulation of the secreted glycoprotein SPARC (also called BM-40 or osteonectin). This protein is a nonstructural component of the extracellular matrix that is thought to regulate cell-matrix interaction during development, wound repair, and carcinogenesis. Its precise function remains unclear. To estimate the contribution of SPARC down-regulation to the major aspects of the transformed phenotype, we have reexpressed this protein from a self-replicating retrovirus Rcas, designated R-SPARC, in the transformed cultures. These R-SPARC-infected cultures display the following main properties: (i) they accumulate the SPARC protein to a level identical to or only slightly higher than the level in normal chick embryo fibroblasts; (ii) they retain the main phenotypic properties characteristic of in vitro transformed cells, that is, altered morphology, capacity to grow in a reduced amount of serum, and capacity to develop colonies from single cells in agar; (iii) they display a clearly reduced capacity to develop local fibrosarcomas in vivo. Taken together, these data strongly suggest that down-regulation of SPARC contributes to the transformed phenotype triggered by vSrc and vJun in primary avian fibroblasts, by facilitating in vivo tumorigenesis.     

Comparative chemosensitivity of circulating human prostate cancer cells and primary cancer cells

The chemosensitivity of circulating PC-3 human prostate cancer cells, isolated from nude mice orthotopically implanted with PC-3, was compared to that of the parental PC-3 cells. PC-3 and circulating PC-3, both labeled with green fluorescent protein (GFP), were seeded in 96-well plates. The MTT assay was then performed at 24, 48, and 72 hours, comparing control cultures to cultures treated with cisplatin at 1, 2.5, 5 and 10 μm/ml, and docetaxel at 10, 20, 25 and 50 μm/ml at each time point. The circulating tumor cells (CTCs) exhibited a significantly increased sensitivity to both cisplatin and docetaxel when compared to PC-3 parental cells, with docetaxel having the greater efficacy. The future goal, based on these studies, is the culture of CTCs from cancer patients' peripheral blood for chemosensitivity testing, for improved individualized therapy.     

Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models

Elucidation of the mechanisms of hormone-independent metastatic prostate cancer remains a significant and highly relevant challenge. We report here that hormone-refractory human prostate carcinoma growing orthotopically efficiently deliver viable metastatic cells in the host circulation. This is in contrast to the ectopic tumors of the same lineage, which do not deliver live cells into the circulation. To investigate the malignant potential of viable circulating carcinoma cells, we developed a novel dual-color orthotopic coimplantation model of human prostate cancer metastasis in nude mice. This model is comprised of coinjection of an equivalent mixture of isolated and cultured circulating green fluorescent protein-expressing clones and parental red fluorescent protein-expressing human prostate carcinoma cells. In the dual-color model, the selected green fluorescent protein-labeled viable circulating cells have an increased metastatic propensity relative to the red fluorescent protein-labeled parental cells. The identification and isolation of highly malignant viable circulating human prostate carcinoma cells from orthotopic but not ectopic models will enable important new insights into the metastatic process including the role of the tumor microenvironment.     

TOPK is highly expressed in circulating tumor cells,enabling metastasis of prostate cancer

Circulating tumor cells (CTCs) are important for metastasis in prostate cancer. T-LAK cell-originated protein kinase (TOPK) is highly expressed in cancer cells. Herein, we established a xenograft animal model, isolated and cultured the CTCs, and found CTCs have significantly greater migratory capacity than parental cells. TOPK is more highly expressed in the CTCs than in parental cells and is also highly expressed in the metastatic nodules caused by CTCs in mice. Knocking down TOPK decreased the migration of CTCs both in vitro and in vivo. TOPK was modulated by the PI3K/PTEN and ERK pathways during the metastasis of prostate cancer. High levels of TOPK in the tumors of patients were correlated with advanced stages of prostate cancer, especially for high-risk patients of Gleason score≥8, PSA>20ng/ml. In summary, TOPK was speculated to be one of a potential marker and therapeutic target in advanced prostate cancer.     

Inhibition of angiogenesis and tumor metastasis by targeting a matrix immobilized cryptic extracellular matrix epitope in laminin

Angiogenesis and tumor metastasis depend on extracellular matrix (ECM) remodeling and subsequent cellular interactions with these modified proteins. An in-depth understanding of how both endothelial and tumor cells use matrix-immobilized cryptic ECM epitopes to regulate invasive cell behavior may lead to the development of novel strategies for the treatment of human tumors. However, little is known concerning the existence and the functional significance of cryptic laminin epitopes in regulating angiogenesis and tumor cell metastasis. Here, we report the isolation and characterization of a synthetic peptide that binds to a cryptic epitope in laminin. The STQ peptide selectively bound denatured and proteolyzed laminin but showed little interaction with native laminin. The cryptic laminin epitope recognized by this peptide was selectively exposed within malignant melanoma in vivo, whereas little if any was detected in normal mouse skin. Moreover, the STQ peptide selectively inhibited endothelial and tumor cell adhesion, migration, and proliferation in vitro and inhibited angiogenesis, tumor growth, and experimental metastasis in vivo. This inhibitory activity was associated with a selective up-regulation of the cyclin-dependent kinase inhibitor P27(KIP1) and induction of cellular senescence. These novel findings suggest the existence of functionally relevant cryptic laminin epitopes in vivo and that selective targeting of these laminin epitopes may represent an effective new strategy for the treatment of malignant tumors by affecting both the endothelial and tumor cell compartments.     

KRAS mutant rectal cancer cells interact with surrounding fibroblasts to deplete the extracellular matrix

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS‐mt) and KRAS‐wild‐type (KRAS‐wt) patients. We found that KRAS‐mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment.     

Inhibition of prostaglandin synthesis and actions by genistein in human prostate cancer cells and by soy isoflavones in prostate cancer patients

Soy and its constituent isoflavone genistein inhibit the development and progression of prostate cancer (PCa). Our study in both cultured cells and PCa patients reveals a novel pathway for the actions of genistein, namely the inhibition of the synthesis and biological actions of prostaglandins (PGs), known stimulators of PCa growth. In the cell culture experiments, genistein decreased cyclooxygenase‐2 (COX‐2) mRNA and protein expression in both human PCa cell lines (LNCaP and PC‐3) and primary prostate epithelial cells and increased 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) mRNA levels in primary prostate cells. As a result genistein significantly reduced the secretion of PGE₂ by these cells. EP4 and FP PG receptor mRNA were also reduced by genistein, providing an additional mechanism for the suppression of PG biological effects. Further, the growth stimulatory effects of both exogenous PGs and endogenous PGs derived from precursor arachidonic acid were attenuated by genistein. We also performed a pilot randomised double blind clinical study in which placebo or soy isoflavone supplements were given to PCa patients in the neo‐adjuvant setting for 2 weeks before prostatectomy. Gene expression changes were measured in the prostatectomy specimens. In PCa patients ingesting isoflavones, we observed significant decreases in prostate COX‐2 mRNA and increases in p21 mRNA. There were significant correlations between COX‐2 mRNA suppression, p21 mRNA stimulation and serum isoflavone levels. We propose that the inhibition of the PG pathway contributes to the beneficial effect of soy isoflavones in PCa chemoprevention and/or treatment. © 2008 Wiley‐Liss, Inc.     

Inhibition of prostate specific antigen expression by genistein in prostate cancer cells

Recent studies have provided convincing evidence for the role of soy-isoflavones, particularly genistein, in the inhibition of prostate cancer cell growth. Prostate specific antigen (PSA) is a biological marker used to detect and monitor the treatment of prostate cancer patients. Previous studies have documented that isoflavones can inhibit the secretion of PSA in the androgen-dependent prostate cancer cell line, LNCaP, however, the effects of genistein on androgen-independent PSA expression has not been explored. In this study, we have utilized a prostate cancer cell line, VeCaP, which expresses PSA in an androgen-independent manner, to determine the effects of genistein on cell proliferation and PSA expression. Here we show that genistein inhibits cell growth similarly in both the LNCaP and VeCaP cell lines, but has differential effects on PSA expression. We demonstrate using concentrations of genistein that have been detected in the serum of humans consuming a soy-rich diet, that genistein decreases PSA mRNA, protein expression and secretion. Conversely, only high concentrations of genistein inhibited PSA expression in VeCaP cells. Additionally, we have demonstrated that genistein inhibits cell proliferation independent of PSA signaling pathways, providing further evidence to support the role of genistein as a chemopreventive/therapeutic agent for prostate cancer irrespective of androgen responsiveness.     

Increased expression of apoptosis inhibitor protein XIAP contributes to anoikis resistance of circulating human prostate cancer metastasis precursor cells

Survival in lymph or blood is an essential prerequisite for metastasis of carcinoma cells to distant organs. Recently, we reported isolation and initial biological characterization of circulating metastatic cells in a fluorescent, orthotopic, metastatic nude-mouse model of human prostate cancer. Here we show that the metastatic human prostate carcinoma cells selected for survival in the circulation have increased resistance to anoikis, which is apoptosis induced by cell detachment. Using gene silencing and gene transfer techniques, we show that increased expression of the apoptosis inhibitory protein XIAP contributes to anoikis resistance of the circulating metastatic human prostate carcinoma cells. We also provide initial preclinical data on the antimetastatic efficacy of recently discovered small-molecule antagonists of XIAP.     

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.     

From transformation to metastasis: deconstructing the extracellular matrix in breast cancer

The extracellular matrix (ECM) is a guiding force that regulates various developmental stages of the breast. In addition to providing structural support for the cells, it mediates epithelial-stromal communication and provides cues for cell survival, proliferation, and differentiation. Perturbations in ECM architecture profoundly influence breast tumor progression and metastasis. Understanding how a dysregulated ECM can facilitate malignant transformation is crucial to designing treatments to effectively target the tumor microenvironment. Here, we address the contribution of ECM mechanics to breast cancer progression, metastasis, and treatment resistance and discuss potential therapeutic strategies targeting the ECM.     

Experimental metastasis of oncogene-transformed NIH 3T3 cells in chick embryo.

By means of a highly sensitive and quantitative assay for specific detection of metastasized tumor cells in chick embryonic organs using the polymerase chain reaction (PCR), we have examined the experimental metastatic ability of individual clones of NIH 3T3 cells, transformed with oncogenes: v-Ki-ras, v-Ha-ras, v-src, v-fos, and v-abl. Such a transformed clone had different metastatic abilities in different embryonic organs. Among them, two clones of NIH 3T3 cells transformed with ras-oncogenes (v-Ki-ras or v-Ha-ras) metastasized to liver and lungs of chick embryo, and grew there more rapidly than the other clones. The parental NIH 3T3 cells were detected as slight bands of PCR products after iv injection, indicating some cells were trapped in chick embryonic organs, but did not grow. These findings indicate that the transformed cells are able to invade the organ tissues and grow in embryonic chick organs, but non-metastatic cells such as the untransformed-NIH 3T3 cells are not able to grow in the secondary sites. These experiments clearly demonstrate the usefulness of this assay system to study genes involved in malignant transformation.     

Inhibition by bovine endothelial cells of degradation by HT-1080 fibrosarcoma cells of extracellular matrix proteins

Medium conditioned by bovine arterial endothelial cells inhibited the degradation by human fibrosarcoma cells of living cultures of rat smooth muscle cells or their cell-free extracellular matrices. Endothelial cell-conditioned medium had no effect on the growth kinetics of fibrosarcoma cells, and the inhibitory influence of conditioned medium on matrix degradation was greatest with low numbers of tumor cells. Conditioned medium inhibited the production of tumor cell plasminogen activators, enzymes previously found to play a role in matrix glycoprotein degradation. The endothelial factor was heat- and acid-stable and non-dialyzable, and mixing experiments showed that it did not directly inactivate the tumor cell plasminogen activator. Endothelial cells may therefore modulate the production of proteolytic enzymes important in the implantation stage of tumor metastasis.     

Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling.

PURPOSE: Generation of a reactive stroma environment occurs in many human cancers and is likely to promote tumorigenesis. However, reactive stroma in human prostate cancer has not been defined. We examined stromal cell phenotype and expression of extracellular matrix components in an effort to define the reactive stroma environment and to determine its ontogeny during prostate cancer progression. EXPERIMENTAL DESIGN: Normal prostate, prostatic intraepithelial neoplasia (PIN), and prostate cancer were examined by immunohistochemistry. Tissue samples included radical prostatectomy specimens, frozen biopsy specimens, and a prostate cancer tissue microarray. A human prostate stromal cell line was used to determine whether transforming growth factor beta1 (TGF-beta1) regulates reactive stroma. RESULTS: Compared with normal prostate tissue, reactive stroma in Gleason 3 prostate cancer showed increased vimentin staining and decreased calponin staining (P < 0.001). Double-label immunohistochemistry revealed that reactive stromal cells were vimentin and smooth muscle alpha-actin positive, indicating the myofibroblast phenotype. In addition, reactive stroma cells exhibited elevated collagen I synthesis and expression of tenascin and fibroblast activation protein. Increased vimentin expression and collagen I synthesis were first observed in activated periacinar fibroblasts adjacent to PIN. Similar to previous observations in prostate cancer, TGF-beta1-staining intensity was elevated in PIN. In vitro, TGF-beta1 stimulated human prostatic fibroblasts to switch to the myofibroblast phenotype and to express tenascin. CONCLUSIONS: The stromal microenvironment in human prostate cancer is altered compared with normal stroma and exhibits features of a wound repair stroma. Reactive stroma is composed of myofibroblasts and fibroblasts stimulated to express extracellular matrix components. Reactive stroma appears to be initiated during PIN and evolve with cancer progression to effectively displace the normal fibromuscular stroma. These studies and others suggest that TGF-beta1 is a candidate regulator of reactive stroma during prostate cancer progression.     

Induction of anchorage independence in human diploid foreskin fibroblasts by carcinogenic metal salts

We studied whether arsenic, nickel, and chromium compounds that are human carcinogens could induce transformation of cultured primary human diploid foreskin cells (HFC). All nickel compounds tested, PbCrO4, K2Cr2O7, CrO3, Na2HAsO4, NaAsO2, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused significant (p = 0.001) dose-dependent inductions of anchorage-independent colonies in HFC. KH2AsO4, CaCl2, MnCl2, and Hg(CH3CO2)2 did not induce anchorage independence. Optimal expression times for induction of anchorage independence in HFC were observed as early as 11 days following treatment with MNNG, Ni3S2, Ni(C2H3O2), or NiSO4. Cell strains derived from anchorage-independent colonies showed 33 to 429-fold higher plating efficiencies in soft agar than parental populations, and the anchorage-independent phenotype was stable for eight passages, at which time cells senesced. Anchorage-independent cell strains derived from metal salt-treated cells were not resistant to the cytotoxicity of metal salts, indicating metal salts induced rather than selected for anchorage independence. Nine of 10 cell strains derived from metal compound- or MNNG-induced anchorage-independent colonies displayed the same or lower saturation densities than untreated human fibroblasts. None of these cell strains escaped senescence or showed definitive morphological transformation. MNNG (1 micrograms/ml) induced anchorage independence and mutation to ouabain resistance and 6-thioguanine resistance in HFC, but concentrations of Ni2S3 that induced anchorage independence did not induce mutation at either locus in HFC. These results demonstrate that carcinogenic metal salts induce stable anchorage independence early in human diploid foreskin fibroblasts, and this anchorage independence is independent of other in vitro markers of fibroblast transformation, such as focus formation or immortality. Metal salt induction of anchorage independence can now be used as an assay to study mechanisms of genotoxicity exerted by carcinogenic metal compounds in human cells.