Sylvia M. Wilson

Ineffectiveness of Doxorubicin Treatment on Solitary Dormant Mammary Carcinoma Cells or Late-developing Metastases

Breast cancer is noted for long periods of tumor dormancy and metastases can occur many years after treatment. Adjuvant chemotherapy is used to prevent metastatic recurrence but is not always successful. As a model for studying mechanisms of dormancy, we have used two murine mammary carcinoma cell lines: D2.0R/R cells, which are poorly metastatic but form metastases in some mice after long latency times, and D2A1/R cells, which form more numerous metastases much earlier. Previously we identified a surprisingly large population of dormant but viable solitary cells, which persisted in an undivided state for up to 11 weeks after injection of D2.0R/R cells. Dormant cells were also detected for D2A1/R cells, in a background of growing metastases. Here we used this model to test the hypothesis that dormant tumor cells would not be killed by cytotoxic chemotherapy that targets actively dividing cells, and that the late development of metastases from D2.0R/R cells would not be inhibited by chemotherapy that effectively inhibited D2A1/R metastases. We injected mice with D2A1/R or D2.0R/R cells via a mesenteric vein to target liver. We developed a doxorubicin (DXR) treatment protocol that effectively reduced the metastatic tumor burden from D2A1/R cells at 3 weeks. However, this treatment did not reduce the numbers of solitary dormant cells in mice injected with either D2A1/R or D2.0R/R cells. Furthermore, DXR did not reduce the metastatic tumor burden after an 11-week latency period in mice injected with D2.0R/R cells. Thus, apparently effective chemotherapy may spare non-dividing cancer cells, and these cells may give rise to metastases at a later date. This study has important clinical implications for patients being treated with cytotoxic chemotherapy.

Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells.

Osteopontin (OPN) has been associated with enhanced malignancy in breast cancer, but its functional role in this disease is poorly understood. To study the effect of OPN on cellular invasiveness, basal OPN expression was first assessed in members of a progression series of human mammary epithelial cell lines (21PT: immortalized, non-tumorigenic; 21NT: weakly tumorigenic; 21MT-1: tumorigenic, weakly metastatic; MDA-MB-435 cells: tumorigenic, highly metastatic). The two lines which expressed lowest basal levels of OPN (21PT, 21NT) were then examined for up-regulation of invasive behavior in response to exogenous or transfected (endogenous) OPN. Both 21PT and 21NT showed increased invasiveness through Matrigel when human recombinant (hr)OPN was added to the lower chamber of transwells. Both also showed a cell migration response to hrOPN. Populations of 21PT and 21NT cells stably transfected with an OPN-expression vector showed higher levels of cell invasiness than control vector transfectants. Examination of transfectants for mRNA of a number of secreted proteases showed that only urokinase-type plasminogen activator (uPA) expression was closely associated with OPN expression and cellular invasiveness. Treatment of the parental 21PT and 21NT cells with exogenous hrOPN resulted in increased uPA mRNA expression and increased urokinase activity of the conditioned media. Both increased cell migration and induction of uPA expression are thus potential mechanisms of increased invasiness of breast epithelial cells in response to OPN.

Osteopontin expression in lung cancer

Osteopontin (OPN), an integrin-binding, transformation-associated protein, is secreted by tumor cell lines in culture and is associated with increased malignancy in some experimental tumor systems. Little is known, however, about the significance of OPN expression in human cancers. The aims of this study were to determine if OPN was expressed in a series of surgically resected lung cancers, and if there was a relationship between OPN expression and clinico-pathologic findings or outcome. Twenty-five patients who underwent curative pulmonary resection were studied prospectively. RNA was extracted from primary tumor and distant normal lung tissue for each patient. OPN RNA levels were evaluated by northern blotting. Immunohistochemistry on paraffin-embedded tissue, using an anti-OPN monoclonal antibody, was performed to assess tissue distribution of OPN protein. OPN RNA and protein were over-expressed in the majority of tumors, relative to paired normal tissue. There was variation in the cells of the tumor that were OPN-immunopositive. In some cases OPN was present in tumor cells, while in the majority of cases OPN was detected primarily in tumor-infiltrating macrophages and necrotic areas. Over-expression of OPN RNA or protein generally was not related to clinico-pathological findings. However, there was a statistically significant association between OPN-immunopositivity in the tumor and patient survival. These findings suggest that OPN levels in lung tumors have the potential to provide clinically important predictive information on patient outcome, and that OPN may play a role in the biology of lung cancer.

Plasma osteopontin : associations with survival and metastasis to bone in men with hormone-refractory prostate carcinoma

Osteopontin (OPN) is a secreted glycoprotein that is detectable in human body fluids. Its increased expression has been found in many malignancies, and a stimulatory effect on human prostate carcinoma cells in vitro has been demonstrated. Plasma OPN levels have been associated with tumor burden and survival in patients with metastatic breast carcinoma. The authors explored these associations in men with hormone‐refractory prostate carcinoma (HRPC).

Serial Plasma Osteopontin Levels Have Prognostic Value in Metastatic Breast Cancer

Purpose: Osteopontin is a malignancy-associated protein measurable in blood and tumor tissue. To evaluate its prognostic value in advanced disease, we conducted a prospective clinical study measuring serial osteopontin plasma levels in women with metastatic breast cancer throughout the course of their disease. Experimental Design: One hundred fifty-eight women with newly diagnosed metastatic breast cancer were enrolled in the study. Plasma osteopontin was measured using our validated ELISA, at baseline and every 3 to 12 weeks during and after therapy until death. Multivariate time-dependent survival analyses were conducted using models that right censored patient outcomes 3, 6, and 12 months after the last known osteopontin measurement. Results: Osteopontin was measured in 1,378 samples (median, 9 per patient). Ninety-nine patients had elevated baseline osteopontin (median, 177 ng/mL; range, 1-2,648 ng/mL). In univariate analysis, elevated baseline osteopontin was associated with short survival (P = 0.02). In a multivariate model incorporating standard prognostic factors, baseline osteopontin was significantly associated with survival duration (relative risk, 1.001; P = 0.038). Metastasis-free interval, visceral metastases, and Eastern Cooperative Oncology Group status 2 to 4 also retained significance. In a multivariate model incorporating standard prognostic factors and changes in sequential osteopontin levels, an osteopontin increase of >250 ng/mL at any time was the variable with the most prognostic value for poor survival (relative risk, 3.26; P = 0.0003), and poor Eastern Cooperative Oncology Group status also retained significance. Conclusions: This is the first study to show that in women with metastatic breast cancer, increases in osteopontin levels over time are strongly associated with poor survival. Sequential monitoring of osteopontin may have use in making treatment decisions for these patients.

Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways

Osteopontin (OPN) is a secreted, integrin-binding glycophosphoprotein that has been implicated in breast cancer. We previously showed that OPN-induced cell migration of mammary epithelial cells (MEC) depends on binding to cell surface integrins and involves activation of the hepatocyte growth factor (HGF) receptor, Met. Here, we show that OPN-induced migration of MEC also requires activation of the epidermal growth factor (EGF) pathway. Synergism was seen between EGF and OPN in inducing cell migration. Furthermore, incubation of cells with exogenous OPN increased ligand (TGFα> EGF) and EGF receptor (EGFR) mRNA expression, as well as EGFR kinase activity. Treatment of cells with anti-TGFα or anti-EGFR antibody, or with tyrphostin-25 (EGFR inhibitor), significantly impaired the cell migration response to OPN. Other more broad-spectrum tyrosine kinase inhibitors and the growth factor/ receptor interaction inhibitor, suramin, also inhibited OPN-induced migration. Using specific signal transduction pathway inhibitors, we have screened for involvement of MEK (MAP kinase kinase), phosphatidylinositol 3-kinase, phospholipase C (PLC), and protein kinase C (PKC). Results implicated all of these pathways in OPN-induced cell migration, the most pronounced effect being seen with PLC and PKC inhibitors. These results suggest that induction of MEC migration by OPN involves a cascade of events including at least two growth factor/receptor pathways and multiple downstream signal transduction pathways. A number of potential targets are thus provided for strategies aimed at blocking the malignancy-promoting effects of OPN.

Dietary Genistein Reduces Metastasis in a Postsurgical Orthotopic Breast Cancer Model

Metastatic spread, not primary tumor burden, is the leading cause of breast cancer deaths. For patient prognosis to improve, new systemic adjuvant therapies that are capable of effectively inhibiting the outgrowth of seeded tumor cells after surgical treatment of the primary breast tumor are needed. To facilitate the preclinical development of such therapies, relevant animal models of breast cancer metastasis that can mimic the postsurgical adjuvant setting are required. Here we developed a preclinical xenograft model of breast cancer metastasis where the primary tumor was removed by surgical resection before systemic adjuvant treatment. We used this model to assess the antimetastatic effect of postsurgical dietary intervention with the soy isoflavone genistein. The anticancer activity of genistein has been established in vitro and in vivo, however, few studies have tested the potential of genistein as an antimetastatic therapy. Using our model, we tested the efficacy of adjuvant treatment with genistein to inhibit the outgrowth of metastases postsurgery. To establish primary tumors, human breast carcinoma cells, MDA-MB-435/HAL, were implanted into the mammary fat pad of female nude mice. Primary tumors were left to grow for 5 weeks before being surgically removed. Mice were then randomized into two diet groups: control soy-free diet versus genistein-supplemented diet. Five weeks later, metastatic burden was assessed. Genistein reduced the percent metastatic burden in the lungs by 10-fold. These results indicate that dietary intervention following cancer surgery can affect the outgrowth of seeded tumor cells. The availability of well-characterized, clinically relevant animal models for studying factors that regulate metastatic outgrowth postsurgery will provide an important tool for developing new systemic adjuvant therapies.

ras mutation and expression of the ras-regulated genes osteopontin and cathepsin L in human esophageal cancer

As part of our ongoing studies to characterize molecular alterations in a well‐defined series of surgically resected esophageal cancers, we examined the expression of 2 ras‐regulated genes, whose products (osteopontin and cathepsin L) previously were shown to be associated with tumor invasion and metastasis. RNA was extracted from primary esophageal tumors (adenocarcinomas, 19; squamous‐cell carcinomas, 6) and matched histologically normal esophageal mucosa from the distant resection margin. Northern analysis was used to quantitate RNA, relative to an 18S rRNA control, and immunohistochemistry to assess the tissue distribution of osteopontin. In addition, H‐, K‐ and N‐ras mutations were studied in the same tissues using PCR and hybridization with allele (mutant)‐specific oligonucleotide probes. We demonstrated a K‐ras mutation (codon 12, GTT) in one esophageal adenocarcinoma. The ras‐regulated gene osteopontin was over‐expressed in 100% of squamous‐cell carcinomas and in 58% of adenocarcinomas relative to matched normal esophageal mucosa. Patterns of immunoreactivity for osteopontin protein also varied between squamous‐cell carcinomas (tumor cell staining) and adenocarcinomas (predominantly tumor‐infiltrating macrophages). Expression of cathepsin L also varied with esophageal tumor histology, with over‐expression in 58% of primary esophageal adenocarcinomas and 33% of squamous‐cell cancers. Int. J. Cancer 72:739–745, 1997.

Up-regulation of TIMP-1 expression in B16-F10 melanoma cells suppresses their metastatic ability in chick embryo

Clonal B16-F10 cell lines with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) have been generated by transfection with a TIMP-1-containing expression vector. The parental B16-F10 and control 1–2 cells, and two TIMP-1 up-regulated clones (2–10, 6–5), were studied for their growth characteristics in tissue culture and their experimental metastatic ability in the chick embryo. Both of the TIMP-1 up-regulated clones showed slower in vitro growth and had lower saturation densities. Both clones were also less metastatic following intravenous injection into chick embryos, and formed significantly fewer metastatic tumors in the chorioallantoic membrane and in the liver than did parental B16-F10 and control cells. Furthermore, the size of tumors formed by TIMP-1 up-regulated cells was significantly reduced in comparison to the tumors produced by B16-F10 or control cells. Our results show that malignant cell lines genetically modified to express increased levels of TIMP-1 exhibit a suppressed experimental metastatic ability in vivo. We propose that TIMP-1 suppresses metastatic ability by decreasing both invasive and growth abilities.

SnoN Is a Cell Type-specific Mediator of Transforming Growth Factor-β Responses

The transforming growth factor-β (TGF-β) family of secreted proteins have pleiotropic functions that are critical to normal development and homeostasis. However, the intracellular mechanisms by which the TGF-β proteins elicit cellular responses remain incompletely understood. The Smad proteins provide a major means for the propagation of the TGF-β signal from the cell surface to the nucleus, where the Smad proteins regulate gene expression leading to TGF-β-dependent cellular responses including the inhibition of cell proliferation. Recent studies have suggested that a nuclear Smad-interacting protein termed SnoN, when overexpressed in cells, suppresses TGF-β-induced Smad signaling and TGF-β inhibition of cell proliferation. However, the physiologic function of endogenous SnoN in TGF-β-mediated biological responses remained to be elucidated. Here, we determined the effect of genetic knock-down of SnoN by RNA interference on TGF-β responses in mammalian cells. Unexpectedly, we found that SnoN knock-down specifically inhibited TGF-β-induced transcription in the lung epithelial cell line Mv1Lu but not in HeLa or HaCaT cells. SnoN knock-down was also found to block TGF-β-dependent cell cycle arrest in Mv1Lu cells. Collectively, these data indicate that rather than suppressing TGF-β-induced responses, endogenous SnoN acts as a positive mediator of TGF-β-induced transcription and cell cycle arrest in lung epithelial cells. Our study also shows that SnoN couples the TGF-β signal to gene expression in a cell-specific manner.