Shijie Sheng

Inhibition of tumor growth and metastasis of human breast cancer cells transfected with tissue inhibitor of metalloproteinase 4

We recently identified, cloned, and characterized a novel human tissue inhibitor of metalloproteinases-4, TIMP-4 (Greene et al., 1996). To determine if TIMP-4 can modulate the in vivo growth of human breast cancers, we transfected a full-length TIMP-4 cDNA into MDA-MB-435 human breast cancer cells and studied the orthotopic growth of TIMP-4-transfected (TIMP4-435) versus control (neo-435) clones in the mammary fat pad of athymic nude mice. TIMP4-435 clones expressed TIMP-4 mRNA and produced anti-metalloproteinase (MMP) activity, while neo-435 clones did not express TIMP-4 mRNA or produce detectable anti-MMP activity. Overexpression of TIMP-4 inhibited the invasion potential of the cells in the in vitro invasion assay. When injected orthotopically into nude mice, TIMP-4 transfectants were significantly inhibited in tumor growth by 4 – 10-fold in primary tumor volumes; and in an axillary lymph node and lung metastasis as compared with controls. These results suggest the therapeutic potential of TIMP-4 in treating cancer malignant progression

Metastasis and AKT activation

Metastasis is responsible for 90% of cancer patient deaths. More information is needed about the molecular basis for its potential detection and treatment. The activated AKT kinase is necessary for many events of the metastatic pathway including escape of cells from the tumors environment, into and then out of the circulation, activation of proliferation, blockage of apoptosis, and activation of angiogenesis. A series of steps leading to metastatic properties can be initiated upon activation of AKT by phosphorylation on Ser-473. These findings lead to the question of how this activation is connected to metastasis. Activated AKT phosphorylates GSK-3β causing its proteolytic removal. This increases stability of the negative transcription factor SNAIL, thereby decreasing transcription of the transmembrane protein E-cadherin that forms adhesions between adjacent cells, thereby permitting their detachment. How is AKT hyperactivated in metastatic cells? Increased PI3K or TORC2 kinase activity- or decreased PHLPP phosphatase could be responsible. Furthermore, a positive feedback mechanism is that the decrease of E-cadherin lowers PTEN and thereby increases PIP3, further activating AKT and metastasis.

Clioquinol, a therapeutic agent for alzheimer's disease, has proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human prostate cancer cells and xenografts

Tumor growth and metastasis depend on angiogenesis that requires the cofactor copper. Consistently, high levels of copper have been found in many types of human cancers, including prostate, breast, colon, and lung. Recent studies suggest that copper could be used as a novel selective target for cancer therapies. Clioquinol is capable of forming stable complexes with copper and currently used in clinics for treatment of Alzheimers disease. Most recently, it has been reported that clioquinol possesses antitumor effects. However, the underlying molecular mechanism is unclear. We report here that after binding to copper, clioquinol can inhibit the proteasomal chymotrypsin-like activity, repress androgen receptor (AR) protein expression, and induce apoptotic cell death in human prostate cancer LNCaP and C4-2B cells. In addition, clioquinol alone exhibits similar effects in prostate cancer cell lines with elevated copper at concentrations similar to those found in patients. Addition of dihydrotestosterone did not affect clioquinol-mediated proteasome inhibition in both prostate cancer cell lines. However, dihydrotestosterone partially inhibited clioquinol-induced AR suppression and apoptosis only in androgen-dependent LNCaP cells. Animal studies show that clioquinol treatment significantly inhibits the growth of human prostate tumor C4-2B xenografts (by 66%), associated with in vivo proteasome inhibition, AR protein repression, angiogenesis suppression, and apoptosis induction. Our study provides strong evidence that clioquinol is able to target tumor proteasome in vivo in a copper-dependent manner, resulting in formation of an active AR inhibitor and apoptosis inducer that is responsible for its observed antiprostate tumor effect.

Maspin expression inhibits osteolysis, tumor growth, and angiogenesis in a model of prostate cancer bone metastasis

Emerging evidence indicates that tumor-associated proteolytic remodeling of bone matrix may underlie the capacity of tumor cells to colonize and survive in the bone microenvironment. Of particular importance, urokinase-type plasminogen activator (uPA) has been shown to correlate with human prostate cancer (PC) metastasis. The importance of this protease may be related to its ability to initiate a proteolytic cascade, leading to the activation of multiple proteases and growth factors. Previously, we showed that maspin, a serine protease inhibitor, specifically inhibits PC-associated uPA and PC cell invasion and motility in vitro. In this article, we showed that maspin-expressing transfectant cells derived from PC cell line DU145 were inhibited in in vitro extracellular matrix and collagen degradation assays. To test the effect of tumor-associated maspin on PC-induced bone matrix remodeling and tumor growth, we injected the maspin-transfected DU145 cells into human fetal bone fragments, which were previously implanted in immunodeficient mice. These studies showed that maspin expression decreased tumor growth, reduced osteolysis, and decreased angiogenesis. Furthermore, the maspin-expressing tumors contained significant fibrosis and collagen staining, and exhibited a more glandular organization. These data represent evidence that maspin inhibits PC-induced bone matrix remodeling and induces PC glandular redifferentiation. These results support our current working hypothesis that maspin exerts its tumor suppressive role, at least in part, by blocking the pericellular uPA system and suggest that maspin may offer an opportunity to improve therapeutic intervention of bone metastasis.

Maspin sensitizes breast carcinoma cells to induced apoptosis

Maspin, a novel serine protease inhibitor (serpin), suppresses the growth and metastasis of breast tumor in vivo. However, the underlying molecular mechanism is unclear. In the current study, we report the first evidence that endogenous maspin expression in mammary carcinoma cells MDA-MB-435 enhanced staurosporine (STS)-induced apoptosis as judged by the increased fragmentation of DNA, increased proteolytic inactivation of poly-[ADP-ribose]-polymerase (PARP), as well as the increased activation of caspase-8 and caspase-3. In parallel, recombinant maspin did not directly regulate the proteolytic activities of either caspase-3 or caspase-8 in vitro. Consistent with this result, maspin expressing normal mammary epithelial cells underwent more rapid STS-induced apoptosis as compared to breast carcinoma cells. Interestingly, maspin transfectant cells did not undergo spontaneous apoptosis in the absence of STS. Moreover, neither purified maspin protein added from outside nor endogenous maspin secreted to the cell culture media sensitized cells to STS-induced apoptosis. To investigate the structural determinants of maspin in its apoptosis-sensitizing effect, MDA-MB-435 cells were also transfected with maspin/PAI-1 and PAI-1/maspin chimeric constructs resulting from swapping the N-terminal and the C-terminal domains between maspin and PAI-1 (plasminogen activator inhibitor type 1). The resulting stable transfectant clones expressing maspin/PAI-1 and PAI-1/maspin, respectively, did not undergo spontaneous apoptosis, and were similarly inhibited as maspin transfectant cells in motility assay. Interestingly, however, expression of both maspin/PAI-1 and PAI-1/maspin in MDA-MB-435 cells failed to sensitize these cells to STS-induced apoptosis. Taken together, our evidence provides new insights into the complex molecular mechanisms of maspin that may suppress breast tumor progression not only at the step of invasion and motility, but also by regulating tumor cell apoptosis. The sensitizing effect of maspin on apoptosis is to be contrasted by the pro-survival effect of several other serpins.

Regulation of FOXO3a/β-catenin/GSK-3β signaling by 3,3'-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells

Previous studies from our laboratory have shown anti-proliferative and pro-apoptotic effects of 3,3′-diindolylmethane (DIM) through regulation of Akt and androgen receptor (AR) in prostate cancer cells. However, the mechanism by which DIM regulates Akt and AR signaling pathways has not been fully investigated. It has been known that FOXO3a and glycogen synthase kinase-3β (GSK-3β), two targets of activated Akt, interact with β-catenin, regulating cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3β, and β-catenin are all AR coregulators and regulate the activity of AR, mediating the development and progression of prostate cancers. Here, we investigated the molecular effects of B-DIM, a formulated DIM with higher bioavailability, on Akt/FOXO3a/GSK-3β/β-catenin/AR signaling in hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We found that B-DIM significantly inhibited the phosphorylation of Akt and FOXO3a and increased the phosphorylation of β-catenin, leading to the inhibition of cell growth and induction of apoptosis. We also found that B-DIM significantly inhibited β-catenin nuclear translocation. By electrophoretic mobility shift and chromatin immunoprecipitation assays, we found that B-DIM inhibited FOXO3a binding to the promoter of AR and promoted FOXO3a binding to the p27KIP1 promoter, resulting in the alteration of AR and p27KIP1 expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive prostate cancer cells. These results suggest that B-DIM-induced cell growth inhibition and apoptosis induction are partly mediated through the regulation of Akt/FOXO3a/GSK-3β/β-catenin/AR signaling. Therefore, B-DIM could be a promising non-toxic agent for possible treatment of hormone-sensitive but most importantly hormone-refractory prostate cancers.

Bax mediates the apoptosis-sensitizing effect of maspin

Maspin, a serine protease inhibitor (serpin), can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. This may occur through maspin-mediated inhibition of pericellular proteolysis. In a recent report, we provided evidence that maspin may also suppress tumor progression by enhancing cellular sensitivity to apoptotic stimuli. To our knowledge, maspin is the only proapoptotic serpin among all of the serpins implicated thus far in apoptosis regulation. The goal of the present study is to identify the specific target molecule(s), the modification of which by maspin renders tumor cells sensitive to chemotherapeutic agents. Our cellular, molecular, and biochemical studies demonstrate an essential role of Bax in the proapoptotic effect of maspin. First, Bax was up-regulated in maspin-transfected prostate and breast tumor cells, whereas the levels of other Bcl-2 family members including Bcl-2, Bcl-xl, and Bak remained unchanged. Second, on apoptosis induction, a greater amount of Bax was translocated from cytosol to mitochondria in maspin-transfected cells. After treatment with a Bax-silencing small interfering RNA, maspin-transfected cells became significantly more resistant to drug-induced apoptosis. Consistently, the release of cytochrome c and Smac/DIABLO from mitochondria was more responsive to apoptosis stimuli in maspin-transfected cells than in the mock-transfected cells. Third, the apoptosis induction of maspin-transfected cells was associated with increased activation of both caspase-8 and caspase-9. However, a caspase-9-specific inhibitor blocked the sensitization effect of maspin in a dose-dependent and time-dependent manner, demonstrating a rate-limiting role for caspase-9. In line with the central role of the Bax-mediated mitochondrial apoptotic pathway, maspin sensitized the apoptotic response of breast and prostate carcinoma cells to various drugs, ranging from death ligands to endoplasmic reticulum stress. The link between maspin and Bax up-regulation explains the loss of maspin-expressing tumor cells in invasive breast and prostate carcinomas. Our data reveal a novel mechanism for tumor suppressive maspin and suggest that maspin may be used as a modifier for apoptosis-based cancer therapy.

Matrix Metalloproteinase Activity and Osteoclasts in Experimental Prostate Cancer Bone Metastasis Tissue

Previously, we and others showed that broad spectrum pharmaceutical inhibition of matrix metalloproteinase (MMP) activity reduces intraosseous tumor burden and bone degradation in animal models of bone metastasis. Herein, we used specific assays to measure net enzymatic activities of individual MMPs during colonization of bone by prostate cancer cells. PC3 cells were injected into the marrow of human fetal femurs previously implanted in SCID mice. Net MMP-9 activity in bone tissues peaked 2 weeks after injection, coinciding with a wave of osteoclast recruitment. In contrast, MMP-2 and MT1-MMP activity did not change. In vitro, co-culture of PC3 cells with bone tissue led to activation of pro-MMP-9 and increases in secreted net MMP-9 activity. Activation of pro-MMP-9 was prevented by metalloprotease inhibitors but not by inhibitors of other classes of proteases. Ribozyme suppression of MMP-9 expression in PC3 cells did not affect pro-MMP-9 activation or net MMP-9 activity and did not affect the phenotype of bone tumors. siRNA targeting of MMP-9 expression in preosteoclasts in vitro demonstrated that tumor-induced preosteoclast motility was dependent on MMP-9 expression. These data suggest that osteoclast-derived MMP-9 may represent a potential therapeutic target in bone metastasis and provide a rationale for the development of MMP-9-specific inhibitors.

Maspin. A tumor suppressing serpin.

Maspin, a serpin found in mammary epithelial cells, has been shown to have tumor suppressor activity. The gene is expressed in normal human mammary epithelial cells but down-regulated in invasive breast carcinomas. Similar patterns of expression at the RNA and protein levels are seen by Northern analysis with cells grown in culture and by immunostaining of tissues. Biological assays of invasion by tumor cells through matrigel membranes and of motility have shown that recombinant maspin inhibits both processes, and that its inhibitory action is totally lost by a single cleavage at the reaction center. Tumor transfectants expressing maspin are inhibited in growth and invasion in nude mice. Maspin is located in the cell membrane and extracellular matrix, and does not behave as a classical inhibitory serpin against any known target protease. Its mode of action is presently unknown.

Tumor suppressive maspin and epithelial homeostasis

Maspin is a 42‐kDa novel serine protease inhibitor (serpin) with multifaceted tumor suppressive activities. To date, the consensus that maspin expression predicts a better prognosis still largely holds for breast, prostate, colon, and oral squamous cancers. Interestingly, however, more detailed analyses revealed a biphasic expression pattern of maspin in early steps of tumorigenesity and re‐expression of maspin in dormant cancer metastastic revertants. These data suggest a sensitivity of maspin expression to changes of epithelial microenvironments, and a role of maspin in epithelial homeostasis. Experimental evidence consistently showed that maspin suppresses tumor growth, invasion and metastasis, induces tumor redifferentiation, and enhances tumor cell sensitivity to apoptosis. Maspin protein isolated from biological sources is a monomer, which is present as a secreted, a cytoplasmic, a nuclear, as well as a cell surface‐associated protein. Nuclear maspin is associated with better prognoses of cancer. It is further noted that extracellular maspin is sufficient to block tumor induced extracellular matrix degradation, tumor cell motility and invasion, whereas intracellular maspin is responsible for the increased cellular sensitivity to apoptosis. Despite these exciting developments, the mechanistic studies of maspin have proven challenging primarily due to the lack of a prototype molecular model. Although the maspin sequence has overall homologies with other members in the serpin superfamily, it does not behave like a typical serpin, that is, non‐inhibitory toward active serine proteases in solution. This novel feature is in line with the X‐ray crystallographic evidence. Several recent studies dedicated to finding the maspin partners support a paradigm shift. The current review is intended to summarize these recent findings and discuss a new perspective of maspin in epithelial homeostasis. J. Cell. Biochem. 97: 651–660, 2006.