Kenneth L. van Golen

Characterization of RhoC Expression in Benign and Malignant Breast Disease: A Potential New Marker for Small Breast Carcinomas with Metastatic Ability

The most important factor in predicting outcome in patients with early breast cancer is the stage of the disease. There is no robust marker capable of identifying invasive carcinomas that despite their small size have a high metastatic potential, and that would benefit from more aggressive treatment. RhoC-GTPase is a member of the Ras-superfamily and is involved in cell polarity and motility. We hypothesized that RhoC expression would be a good marker to identify breast cancer patients with high risk of developing metastases, and that it would be a prognostic marker useful in the clinic. We developed a specific anti-RhoC antibody and studied archival breast tissues that comprise a broad spectrum of breast disease. One hundred eighty-two specimens from 164 patients were used. Immunohistochemistry was performed on formalin-fixed tissues. Staining intensity was graded 0 to 3+ (0 to 1+ was considered negative and 2 to 3+ was considered positive). RhoC was not expressed in any of the normal, fibrocystic changes, atypical hyperplasia, or ductal carcinoma in situ, but was expressed in 36 of 118 invasive carcinomas and strongly correlated with tumor stage (P = 0.01). RhoC had high specificity (88%) in detecting invasive carcinomas with metastatic potential. Of the invasive carcinomas smaller than 1 cm, RhoC was highly specific in detecting tumors that developed metastases. RhoC expression was associated with negative progesterone receptor and HER-2/neu overexpression. We characterized RhoC expression in human breast tissues. RhoC is specifically expressed in invasive breast carcinomas capable of metastasizing, and it may be clinically useful in patients with tumors smaller than 1 cm to guide treatment.

Molecular biology of breast cancer metastasis Inflammatory breast cancer: clinical syndrome and molecular determinants

Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer (LABC) that effects approximately 5% of women with breast cancer annually in the USA. It is a clinically and pathologically distinct form of LABC that is particularly fast growing, invasive, and angiogenic. Nearly all women have lymph node involvement at the time of diagnosis, and approximately 36% have gross distant metastases. Despite recent advances in multimodality treatments, the prognosis of patients with IBC is poor, with a median disease-free survival of less than 2.5 years. Recent work on the genetic determinants that underlie the IBC phenotype has led to the identification of genes that are involved in the development and progression of this disease. This work has been aided by the establishment of primary human cell lines and animal models. These advances suggest novel targets for future interventions in the diagnosis and treatment of IBC.

S100P Promotes Pancreatic Cancer Growth, Survival, and Invasion

Purpose: In the current study, we examined the functional significance and mechanism of action of S100P in pancreatic cancer cells. Experimental Design: S100P levels were increased in Panc-1 cells, which do not express S100P, by transfection with an S100P cDNA and S100P levels were reduced in BxPC3 cells, which express high levels of S100P, by small interfering RNA gene silencing. Effects of these manipulations on cell proliferation, resistance to apoptotic insults, cell migration, and invasion were estimated in vitro using standard assays. The influences of S100P on tumor growth in vivo were studied using xenograft mouse models. To identify the mechanisms involved in these responses, coimmunoprecipitation studies were conducted with S100P with receptor for advanced glycation end products (RAGE) and the effects of inhibiting RAGE using an antagonistic peptide were analyzed. Results: S100P levels correlated with the rates of cell proliferation, survival, migration, and invasion in both cell models in vitro. In vivo, increased S100P levels increased the growth of tumors in mice with s.c.-implanted Panc-1 cells and decreased S100P levels decreased tumor growth after orthotopic implantation of BxPC-3 cells. A direct interaction between S100P and RAGE was indicated by coimmunoprecipitation of these molecules from pancreatic cancer cells. A RAGE antagonist peptide inhibited this interaction and also inhibited the biological effects of S100P on these cells in vitro. Conclusions: These data suggest that S100P plays a major role in the aggressiveness of pancreatic cancer that is likely mediated by its ability to activate RAGE. Thus, interference with S100P may provide a novel approach for treatment of pancreatic cancer.

Stepping out of the flow: capillary extravasation in cancer metastasis.

In order for cancer cells to successfully colonize a metastatic site, they must detach from the primary tumor using extracellular matrix-degrading proteases, intravasate and survive in the circulation, evade the immune response, and extravasate the vasculature to invade the target tissue parenchyma, where metastatic foci are established. Though many of the steps of metastasis are widely studied, the precise cellular interactions and molecular alterations associated with extravasation are unknown, and further study is needed to elucidate the mechanisms inherent to this process. Studies of leukocytes localized to inflamed tissue during the immune response may be used to elucidate the process of cancer extravasation, since leukocyte diapedesis through the vasculature involves critical adhesive interactions with endothelial cells, and both leukocytes and cancer cells express similar surface receptors capable of binding endothelial adhesion molecules. Thus, leukocyte extravasation during the inflammatory response has provided a model for transendothelial migration (TEM) of cancer cells. Leukocyte extravasation is characterized by a process whereby rolling mediated by cytokine-activated endothelial selectins is followed by firmer adhesions with β1 and β2 integrin subunits to an activated endothelium and subsequent diapedesis, which most likely involves activation of Rho GTPases, regulators of cytoskeletal rearrangements and motility. It is controversial whether such selectin-mediated rolling is necessary for TEM of cancer cells. However, it has been established that similar stable adhesions between tumor and endothelial cells precede cancer cell transmigration through the endothelium. Additionally, there is support for the preferential attachment of tumor cells to the endothelium and, accordingly, site-specific metastasis of cancer cells. Rho GTPases are critical to TEM of cancer cells as well, and some progress has been made in understanding the specific roles of the Rho GTPase family, though much is still unknown. As the mechanisms of cancer TEM are elucidated, new approaches to study and target metastasis may be utilized and developed.

Persistent E-Cadherin Expression in Inflammatory Breast Cancer

E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion. Because loss of E-cadherin expression results in disruption of cellular clusters, it has been postulated that E-cadherin functions as a tumor suppressor protein. The role of E-cadherin in inflammatory breast cancer (IBC), a distinct and highly aggressive form of breast cancer, is largely unknown. The aim of our study was to elucidate whether E-cadherin expression contributes to the development and progression of the IBC phenotype and to investigate any differences in E-cadherin expression between IBC and stage-matched non-IBC. Forty-two breast cancer cases (20 IBC and 22 non-IBC) were identified. Strict and well-accepted criteria were used for the diagnosis of IBC. Clinical and pathologic features were studied, and formalin-fixed, paraffin-embedded tissue sections were immunostained for E-cadherin, estrogen and progesterone receptors (ER and PR, respectively), and HER2/neu. Statistical analysis was performed using Fishers exact test. All IBC uniformly expressed E-cadherin, whereas 15 of the 22 (68%) of the non-IBC expressed the protein (P = .006). Intralymphatic tumor emboli in the IBC cases were also all E-cadherin positive. Two IBC tumors demonstrated invasive lobular histology, and both cases were positive for E-cadherin. Of the non-IBC cases, three were invasive lobular carcinomas, and all were positive for E-cadherin. No association was found between E-cadherin expression and ER, PR status, or HER2/neu overexpression. Our study demonstrates that there is a strong association between E-cadherin expression and IBC and suggests that E-cadherin may be involved in the pathogenesis of this form of advanced breast cancer. In our study, we demonstrate that circulating IBC tumor cells strongly express E-cadherin, thereby providing an important exception to the positive association between E-cadherin loss and poor prognosis in breast cancer.

WISP3 is a novel tumor suppressor gene of inflammatory breast cancer

Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a 5-year disease-free survival of less than 45%. Little is known about the genetic alterations that result in IBC. In our previous work, we found that WISP3 was specifically lost in human IBC tumors when compared to stage-matched, non-IBC tumors. We hypothesize that WISP3 has tumor suppressor function in the breast and that it may be a key genetic alteration that contributes to the unique IBC phenotype. The full-length WISP3 cDNA was sequenced and cloned into an expression vector. The resulting construct was introduced in to the SUM149 cell line that was derived from a patient with IBC and lacks WISP3 expression. In soft agar, stable WISP3 transfectants formed significantly fewer colonies than the controls. Stable WISP3 transfectants lost their ability to invade and had reduced angiogenic potential. WISP3 transfection was effective in suppressing in vivo tumor growth in nude mice. Mice bearing WISP3 expressing tumors had a significantly longer survival than those with vector-control transfectant tumors. Our data demonstrate that WISP3 acts as a tumor suppressor gene in the breast. Loss of WISP3 expression contributes to the phenotype of IBC by regulating tumor cell growth, invasion and angiogenesis.

Type I collagen receptor (α2β1) signaling promotes the growth of human prostate cancer cells within the bone

The most frequent site of prostate cancer metastasis is the bone. Adhesion to bone-specific factors may facilitate the selective metastasis of prostate cancer to the skeleton. Therefore, we tested whether prostate cancer bone metastasis is mediated by binding to type I collagen, the most abundant bone protein. We observed that only bone metastatic prostate cancer cells bound collagen I, whereas cells that form only visceral metastases failed to bind collagen. To confirm the relationship between collagen adhesion and bone metastatic potential, a collagen-binding variant of human LNCaP prostate cancer cells was derived through serial passage on type I collagen (LNCaPcol). Fluorescence-activated cell sorting analysis showed that LNCaPcol cells express increased levels of the integrin collagen I receptor α2β1 compared with LNCaP cells. Antibodies to the α2β1 complex inhibited LNCaPcol binding to collagen, confirming that integrins mediated the attachment. Correspondingly, LNCaPcol cells displayed enhanced chemotactic migration toward collagen I compared with LNCaP cells, an activity that could be blocked with α2β1 antibodies. To directly test the role of α2β1-dependent collagen binding in bone metastasis, LNCaP and LNCaPcol cells were injected into the tibia of nude mice. After 9 weeks, 7 of 13 (53%) mice injected with LNCaPcol developed bone tumors, whereas 0 of 8 mice injected with LNCaP cells had evidence of boney lesions. LNCaPcol cells were found to express increased levels of the metastasis-promoting RhoC GTPase compared with parental LNCaP. We conclude that collagen I attachment mediated by α2β1 initiates motility programs through RhoC and suggest a mechanism for prostate cancer metastasis to the bone. (Cancer Res 2006; 66(17): 8648-54)

Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer

Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer known. IBC carries a guarded prognosis primarily due to rapid onset of disease, typically within six months, and the propensity of tumor emboli to invade the dermal lymphatics and spread systemically. Although the clinical manifestations of IBC have been well documented, until recently little was known about the genetic mechanisms underlying the disease. In a comprehensive study aimed at identifying the molecular mechanisms responsible for the unique IBC phenotype, our laboratory identified overexpression of RhoC GTPase in over 90% of IBC tumors in contrast to 36% of stage-matched non-IBC tumors. We also demonstrated that overexpression of RhoC GTPase in human mammary epithelial (HME) cells nearly recapitulated the IBC phenotype with regards to invasion, motility and angiogenesis. In the current study we sought to delineate which signaling pathways were responsible for each aspect of the IBC phenotype. Using well-established inhibitors to the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways. We found that activation of the MAPK pathway was responsible for motility, invasion and production of angiogenic factors. In contrast, growth under anchorage independent conditions was dependent on the PI3K pathway.

Angiogenesis, lymphangiogenesis, growth pattern, and tumor emboli in inflammatory breast cancer: a review of the current knowledge.

This objective of the current review was to provide the reader with a comprehensive summary of the literature related to 3 important and inter‐related features of the biology of inflammatory breast cancer (IBC): angiogenesis, lymphangiogenesis, and the formation of tumor emboli. Information derived from animal models of IBC as well as from translational studies using tissue samples of patients with IBC are discussed. Cancer 2010;116(11 suppl):2748–54.

Rho-Regulatory Proteins in Breast Cancer Cell Motility and Invasion

The importance of the Rho-GTPases in cancer progression, particularly in the area of metastasis, is becoming increasingly evident. This review will provide an overview of the role of the Rho-regulatory proteins in breast cancer metastatis.