Melanie A. Simpson

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-α4β1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.

Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.

Hyaluronan (HA), a secreted glycosaminoglycan component of extracellular matrices, is critical for cellular proliferation and motility during development. However, elevated circulating and cell-associated levels correlate with various types of cancer, including prostate. We have previously shown that aggressive PC3M-LN4 prostate tumor cells synthesize excessive HA relative to less aggressive cells, and express correspondingly higher levels of the HA biosynthetic enzymes HAS2 and HAS3. Inhibition of these enzymes by stable transfection of PC3M-LN4 cells with anti-sense HAS2 or HAS3 expression constructs diminishes HA synthesis and surface retention. In this report, we used these HA-deficient cell lines to examine the role of HA in tumorigenicity. Subcutaneous injection of SCID mice with hyaluronan synthase (HAS) antisense-transfected cells produced tumors threefold to fourfold smaller than control transfectants. Tumors from HAS antisense transfectants were histologically HA-deficient relative to controls. HA deficiency corresponded to threefold reduced cell numbers per tumor, but comparable numbers of apoptotic and proliferative cells. Percentages of apoptotic cells in cultured transfectants were identical to those of control cells, but antisense inhibition of HA synthesis effected slower growth rate of cells in culture. Quantification of blood vessel density within tumor sections revealed 70 to 80% diminished vascularity of HAS antisense tumors. Collectively, the results suggest HAS overexpression by prostate tumor cells may facilitate their growth and proliferation in a complex environment by enhancing intrinsic cell growth rates and promoting angiogenesis. Furthermore, this is the first report of a role for inhibition of HA synthesis in reducing tumor growth kinetics.

Manipulation of Hyaluronan Synthase Expression in Prostate Adenocarcinoma Cells Alters Pericellular Matrix Retention and Adhesion to Bone Marrow Endothelial Cells

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with α4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.

Spontaneous Metastasis of Prostate Cancer Is Promoted by Excess Hyaluronan Synthesis and Processing

Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment.

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells.

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interactionin vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70–90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15–25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.

Hyaluronan facilitates invasion of colon carcinoma cells in vitro via interaction with CD44.

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.

Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models

Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. This characteristic can be exploited for optical imaging of tumors in mice. A near-infrared fluorophore, IRDye 800CW, emission maximum 794 nm, was conjugated to 2-deoxyglucose (2-DG). An immunofluorescent cell-based assay was used to evaluate specificity and sensitivity of the conjugate in cultured cell monolayers. Dose-dependent uptake was established with increasing concentrations of IRDye 800CW 2-DG for epithelial and prostate carcinomas. IRDye 800CW 2-DG was specifically blocked by an antibody against GLUT1 glucose transporter, and by excess unlabeled 2-DG or d-glucose. Signal was increased by a phorbol ester activator of glucose transport. Fluorescence microscopy data confirmed localization of the conjugate in the cytoplasm. Subsequent in vivo studies optimized dose, clearance, and timing for signal capture in nude mouse xenografts. In all cases, tumors were clearly imaged with good signal-to-noise characteristics. These data indicate that IRDye 800CW 2-DG is a broadly applicable optical imaging agent for in vivo imaging of neoplasms in mice.

Hyaluronan and hyaluronidase in genitourinary tumors.

Genitourinary cancers are the most frequently diagnosed cancers in men and the fifth most common in women. Management of disease through accurate and cost effective early diagnostic markers, as well as identification of valid prognostic indicators, has contributed significantly to improved treatment outcomes. In this review, we will discuss the function, regulation and clinical utility of hyaluronan (HA), genes encoding its metabolic enzymes and receptors that mediate its cellular effects. Specific HA synthase (HAS) and hyaluronidase (HAase) genes encode the enzymes that produce HA polymers and oligosaccharides, respectively. Differential effects of these enzymes in progression of genitourinary tumors are determined by the relative balance between HAS and HAase levels, as well as the distribution of receptors. The genes are regulated in a complex fashion at the transcriptional and post-translational levels, but also by epigenetic events, alternative mRNA splicing, and subcellular localization. Importantly, the major tumor-derived HAase enzyme, HYAL-1, either alone or together with HA, is an accurate diagnostic and prognostic marker for genitourinary tumors.

Hyaluronan synthase-3 is upregulated in metastatic colon carcinoma cells and manipulation of expression alters matrix retention and cellular growth.

HA is a glycosaminoglycan that is synthesized on the inner surface of the plasma membrane and secreted into the pericellular matrix. HA and its biosynthetic enzymes (HAS1, HAS2 and HAS3) are thought to participate in tumor growth and cancer progression. In our study, colon carcinoma cells isolated from a lymph node metastasis (SW620) produced more pericellular HA and expressed higher levels of HAS3 mRNA compared to cells isolated from a primary colon carcinoma (SW480). To assess functionality, HAS3 expression in SW620 cells was inhibited by transfection with an asHAS3 construct. Decreased HA secretion and cell‐surface retention by asHAS3 transfectants were confirmed using competitive binding and particle exclusion assays. Anchorage‐independent growth, a correlate of tumor growth in vivo, was assessed by colony formation in soft agar. SW620 cells stably transfected with asHAS3 demonstrated significant growth inhibition, as evidenced by fewer colonies and smaller colony area than either SW620 cells or cells transfected with vector alone. Addition of exogenous HA restored growth in asHAS3 transfectants. Thus, we demonstrate that pericellular HA secretion and retention and HAS3 expression are increased in metastatic colon carcinoma cells relative to cells derived from a primary tumor. Inhibition of HAS3 expression in these cells decreased the pericellular HA matrix and inhibited anchorage‐independent growth. These data suggest that HA and HAS3 function in the growth and progression of colon carcinoma.