Steve Goodison

The RhoGAP Protein DLC-1 Functions as a Metastasis Suppressor in Breast Cancer Cells

The identification of molecular signatures characteristic of tumor cells that are capable of metastatic spread is required for the development of therapeutic interventions to abrogate this lethal process. To facilitate this, we have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 are spontaneously occurring sublines of the MDA-MB-435 cell breast tumor cell line that exhibit phenotypic differences in growth, invasion, and metastatic efficiency in athymic mice. In this study, transcriptional profiles of these cell lines were created using oligonucleotide microarrays representing over 12,000 genes. Intensity modeling and hierarchical clustering analysis identified a 171-gene expression signature that correlated with metastatic phenotype and highlighted several GTPase signaling components. Restoration of one of these GTPases, deleted in liver cancer-1 (DLC-1), in metastatic M4A4 cells to levels observed in the nonmetastatic NM2C5 cell line resulted in the inhibition of migration and invasion in vitro and a significant reduction in the ability of these cells to form pulmonary metastases in athymic mice. These studies show the utility of expression profiling, in an appropriate experimental system, to identify genetic determinants of metastatic sufficiency. The finding that DLC-1 can act as a metastasis-suppressor gene supports an influential role for GTPase signaling in tumor progression.

Improved breast cancer prognosis through the combination of clinical and genetic markers

MOTIVATION Accurate prognosis of breast cancer can spare a significant number of breast cancer patients from receiving unnecessary adjuvant systemic treatment and its related expensive medical costs. Recent studies have demonstrated the potential value of gene expression signatures in assessing the risk of post-surgical disease recurrence. However, these studies all attempt to develop genetic marker-based prognostic systems to replace the existing clinical criteria, while ignoring the rich information contained in established clinical markers. Given the complexity of breast cancer prognosis, a more practical strategy would be to utilize both clinical and genetic marker information that may be complementary. METHODS A computational study is performed on publicly available microarray data, which has spawned a 70-gene prognostic signature. The recently proposed I-RELIEF algorithm is used to identify a hybrid signature through the combination of both genetic and clinical markers. A rigorous experimental protocol is used to estimate the prognostic performance of the hybrid signature and other prognostic approaches. Survival data analyses is performed to compare different prognostic approaches. RESULTS The hybrid signature performs significantly better than other methods, including the 70-gene signature, clinical makers alone and the St. Gallen consensus criterion. At the 90% sensitivity level, the hybrid signature achieves 67% specificity, as compared to 47% for the 70-gene signature and 48% for the clinical makers. The odds ratio of the hybrid signature for developing distant metastases within five years between the patients with a good prognosis signature and the patients with a bad prognosis is 21.0 (95% CI:6.5-68.3), far higher than either genetic or clinical markers alone. AVAILABILITY The breast cancer dataset is available at www.nature.com and Matlab codes are available upon request.

Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion

IntroductionThe biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising.MethodsHuman tissue samples of invasive breast cancer and normal breast, as well as breast cancer cell lines, were evaluated for protein kinase D (PKD) expression, to test if altered expression could serve as a marker for invasive breast cancer. We further utilised specific PKD1-shRNA and a system to inducibly-express PKD1 to analyse the role of PKD1 in the invasive behaviour of breast cancer cell lines in two-dimensional (2D) and three-dimensional (3D) culture. Invasive behaviour in breast cancer cell lines has been linked to matrix metalloproteinases (MMPs), so we also determined if PKD1 regulates the expression and activity of these enzymes.ResultsWe found that the serine/threonine kinase, PKD1, is highly expressed in ductal epithelial cells of normal human breast tissue, but is reduced in its expression in more than 95% of all analysed samples of human invasive breast tumours. Additionally, PKD1 is not expressed in highly invasive breast cancer cell lines, whereas non-invasive or very low-invasive breast cancer cell lines express PKD1. Our results further implicate that in MDA-MB-231 cells PKD1 expression is blocked by epigenetic silencing via DNA methylation. The re-expression of constitutively-active PKD1 in MDA-MB-231 cells drastically reduced their ability to invade in 2D and 3D cell culture. Moreover, MCF-7 cells acquired the ability to invade in 2D and 3D cell culture when PKD1 expression was knocked-down by shRNA. PKD1 also regulated the expression of breast cancer cell MMPs, MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, providing a potential mechanism for PKD1 mediation of the invasive phenotype.ConclusionsOur results identify decreased expression of the PKD1 as a marker for invasive breast cancer. They further suggest that the loss of PKD1 expression increases the malignant potential of breast cancer cells. This may be due to the function of PKD1 as a negative regulator of MMP expression. Our data suggest re-expression of PKD1 as a potential therapeutic strategy.

Persistent Exposure to Mycoplasma Induces Malignant Transformation of Human Prostate Cells

Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including those of the prostate. The American Cancer Society, estimates that approximately 20% of all worldwide cancers are caused by infection. Mycoplasma, a genus of bacteria that lack a cell wall, are among the few prokaryotes that can grow in close relationship with mammalian cells, often without any apparent pathology, for extended periods of time. In this study, the capacity of Mycoplasma genitalium, a prevalent sexually transmitted infection, and Mycoplasma hyorhinis, a mycoplasma found at unusually high frequency among patients with AIDS, to induce a malignant phenotype in benign human prostate cells (BPH-1) was evaluated using a series of in vitro and in vivo assays. After 19 weeks of culture, infected BPH-1 cells achieved anchorage-independent growth and increased migration and invasion. Malignant transformation of infected BPH-1 cells was confirmed by the formation of xenograft tumors in athymic mice. Associated with these changes was an increase in karyotypic entropy, evident by the accumulation of chromosomal aberrations and polysomy. This is the first report describing the capacity of M. genitalium or M. hyorhinis infection to lead to the malignant transformation of benign human epithelial cells and may serve as a model to further study the relationship between prostatitis and prostatic carcinogenesis.

Effects of CXCR4 antagonist CTCE-9908 on prostate tumor growth†

Recent reports have linked the survival‐promoting effect of CXCR4 to the up regulation of Bcl‐2 protein expression.

An Invasion-Independent Pathway of Blood-Borne Metastasis: A New Murine Mammary Tumor Model

It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.

Enrichment of Glycoproteins Using Nanoscale Chelating Concanavalin A Monolithic Capillary Chromatography

Immobilized lectin chromatography can be employed for glycoprotein enrichment, but commonly used columns have limitations of yield and resolution. To improve efficiency and to make the technique applicable to minimal sample material, we have developed a nanoscale chelating Concanavalin A (Con A) monolithic capillary prepared using GMA-EDMA (glycidyl methacrylate-co-ethylene dimethacrylate) as polymeric support. Con A was immobilized on Cu(II)-charged iminodiacetic acid (IDA) regenerable sorbents by forming a IDA:Cu(II):Con A sandwich affinity structure that has high column capacity, as well as stability. When compared with conventional Con A lectin chromatography, the monolithic capillary enabled the better reproducible detection of over double the number of unique N-glycoproteins in human urine samples. Utility for analysis of minimal biological samples was confirmed by the successful elucidation of glycoprotein profiles in mouse urine samples at the microliter scale. The improved efficiency of the nanoscale monolithic capillary will impact the analysis of glycoproteins in complex biological samples, especially where only limited material may be available.

Bladder Cancer–Associated Gene Expression Signatures Identified by Profiling of Exfoliated Urothelia

Bladder cancer is the fifth most commonly diagnosed malignancy in the United States and one of the most prevalent worldwide. It harbors a probability of recurrence of >50%; thus, rigorous, long-term surveillance of patients is advocated. Flexible cystoscopy coupled with voided urine cytology is the primary diagnostic approach, but cystoscopy is an uncomfortable, invasive procedure and the sensitivity of voided urine cytology is poor in all but high-grade tumors. Thus, improvements in noninvasive urinalysis assessment strategies would benefit patients. We applied gene expression microarray analysis to exfoliated urothelia recovered from bladder washes obtained prospectively from 46 patients with subsequently confirmed presence or absence of bladder cancer. Data from microarrays containing 56,000 targets was subjected to a panel of statistical analyses to identify bladder cancer-associated gene signatures. Hierarchical clustering and supervised learning algorithms were used to classify samples on the basis of tumor burden. A differentially expressed geneset of 319 gene probes was associated with the presence of bladder cancer (P < 0.01), and visualization of protein interaction networks revealed vascular endothelial growth factor and angiotensinogen as pivotal factors in tumor cells. Supervised machine learning and a cross-validation approach were used to build a 14-gene molecular classifier that was able to classify patients with and without bladder cancer with an overall accuracy of 76%. Our results show that it is possible to achieve the detection of bladder cancer using molecular signatures present in exfoliated tumor urothelia. Further investigation and validation of the cancer-associated profiles may reveal important biomarkers for the noninvasive detection and surveillance of bladder cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(2):444–53)

Membrane Glycoproteins Associated with Breast Tumor Cell Progression Identified by a Lectin Affinity Approach

The membrane glycoprotein component of the cellular proteome represents a promising source for potential disease biomarkers and therapeutic targets. Here we describe the development of a method that facilitates the analysis of membrane glycoproteins and apply it to the differential analysis of breast tumor cells with distinct malignant phenotypes. The approach combines two membrane extraction procedures, and enrichment using ConA and WGA lectin affinity columns, prior to digestion and analysis by LC-MS/MS. The glycoproteins are identified and quantified by spectral counting. Although the distribution of glycoprotein expression as a function of MW and p I was very similar between the two related cell lines tested, the approach enabled the identification of several distinct membrane glycoproteins with an expression index correlated with either a precancerous (MCF10AT1), or a malignant, metastatic cellular phenotype (MCF10CA1a). Among the proteins associated with the malignant phenotype, Gamma-glutamyl hydrolase, CD44, Galectin-3-binding protein, and Syndecan-1 protein have been reported as potential biomarkers of breast cancer.

Morphological evidence for an invasion-independent metastasis pathway exists in multiple human cancers

BackgroundWe have previously described an alternative invasion-independent pathway of cancer metastasis in a murine mammary tumor model. This pathway is initiated by intravasation of tumor nests enveloped by endothelial cells of sinusoidal vasculature within the tumor. In this study, we examined whether evidence for the invasion-independent pathway of metastasis is present in human cancers.MethodsArchival specimens of 10 common types of human cancers were examined for the presence of sinusoidal vasculature enveloping tumor nests and subsequently generated endothelial-covered tumor emboli in efferent veins.ResultsA percentage of tumor emboli in all cancers was found to be enveloped by endothelial cells, but these structures were particularly prevalent in renal cell carcinomas, hepatocellular carcinomas and follicular thyroid carcinomas. A common feature of the vasculature in these tumors was the presence of dilated sinusoid-like structures surrounding tumor nests. A high mean vascular area within tumors, an indication of sinusoidal vascular development, was significantly related to the presence of endothelial-covered tumor emboli.ConclusionsThese results suggest that an invasion-independent metastatic pathway is possible in a wide variety of human cancers. Further investigation of this phenomenon may present new therapeutic strategies for the amelioration of cancer metastasis.