Zhi Fen Wu

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.

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.

WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. We found concordant and consistent alterations of two genes in 90% of IBC tumors when compared with stage-matched non-IBC tumors: overexpression of RhoC guanosine triphosphatase and loss of WNT-1 induced secreted protein 3 (WISP3). Further work revealed that RhoC is a transforming oncogene for human mammary epithelial (HME) cells. Despite the aggressiveness of the RhoC-driven phenotype, it does not quantitatively reach that of the true IBC tumors. We have demonstrated that WISP3 has tumor growth and angiogenesis inhibitory functions in IBC. We proposed that RhoC and WISP3 cooperate in the development of IBC.MethodsUsing an antisense approach, we blocked WISP3 expression in HME cells. Cellular proliferation and growth were determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and anchorage-independent growth in a soft agar assay. Vascular endothelial growth factor (VEGF) was measured in conditioned medium by enzyme-linked immunosorbent assay.ResultsAntisense inhibition of WISP3 in HME cells increased RhoC mRNA levels and resulted in an increase in cellular proliferation, anchorage-independent growth and VEGF levels in the conditioned medium. Conversely, restoration of WISP3 expression in the highly malignant IBC cell line SUM149 was able to decrease the expression of RhoC protein.ConclusionWISP3 modulates RhoC expression in HME cells and in the IBC cell line SUM149. This provides further evidence that these two genes act in concert to give rise to the highly aggressive IBC phenotype. We propose a model of this interaction as a starting point for further investigations.

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.

RhoC Induces Differential Expression of Genes Involved in Invasion and Metastasis in MCF10A Breast Cells

Inflammatory breast cancer (IBC) is the most deadly form of breast cancer in humans presumably due to its ability to metastasize from its inception. In our laboratory, overexpression of RhoC GTPase was observed to be specific for IBC tumors, but not for stage-matched, non-IBC tumors. RhoC is known to contribute to an IBC-like phenotype in HPV-E6E7 immortalized breast cells. To further study the effect of RhoC overexpression on IBC metastasis, we generated stable transfectants of spontaneous immortalized mammary epithelial cells (MCF10A) overexpressing wild-type RhoC or a constitutively active RhoC mutant (G14V). Both the RhoC wild type and the G14V transfectants were highly invasive and proliferated more rapidly compared to vector-only control clones. Overexpression of RhoC led to an increase in actin stress fiber and focal adhesion contact formation. Comparative microarray analysis of these clones further revealed that RhoC overexpression upregulated 108 genes whereas seven genes were down-regulated. We have further verified by quantitative RT-PCR that genes involved in cell proliferation, invasion/adhesion, and angiogenesis were modulated by RhoC. This work suggests strong candidates for the downstream oncogenic functions of RhoC.

Pomegranate Fruit Extract Impairs Invasion and Motility in Human Breast Cancer

Purpose. Pomegranate fruit extracts (PFEs) possess polyphenolic and other compounds with antiproliferative, pro-apoptotic and anti-inflammatory effects in prostate, lung, and other cancers. Because nuclear transcription factor-kB (NF-kB) is known to regulate cell survival, proliferation, tumorigenesis, and inflammation, it was postulated that PFEs may exert anticancer effects at least in part by modulating NF-kB activity. Experimental design. The authors investigated the effect of a novel, defined PFE consisting of both fermented juice and seed oil on the NF-kB pathway, which is constitutively active in aggressive breast cancer cell lines. The effects of the PFE on NF-kB—regulated cellular processes such as cell survival, proliferation, and invasion were also examined. Results. Analytical characterization of the bioactive components of the PFE revealed active constituents, mainly ellagitannins and phenolic acids in the aqueous PFE and conjugated octadecatrienoic acids in the lipid PFE derived from seeds.The aqueous PFE dose-dependently inhibited NF-kB—dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while decreasing RhoC and RhoA protein expression. Conclusion. Inhibition of motility and invasion by PFEs, coincident with suppressed RhoC and RhoA protein expression, suggests a role for these defined extracts in lowering the metastatic potential of aggressive breast cancer species.

Role of Smad3 in the regulation of rat telomerase reverse transcriptase by TGFβ

Telomerase is induced in certain pathological conditions such as cancer and tissue injury and repair. This induction in fibroblasts from injured lung is repressed by transforming growth factor β (TGFβ) via yet unknown mechanisms. In this study, the role of Smad3 in the inhibition of telomerase reverse transcriptase (TERT) gene transcription by TGFβ was investigated. The rat TERT (rTERT) gene promoter was cloned by PCR amplification and fused with a luciferase reporter gene. This construct was used to analyse regulation of promoter activity in fibroblasts isolated from bleomycin-injured lung with induced telomerase activity. The results showed that TGFβ inhibited rTERT transcription while stimulating Smad3 expression. Interestingly, TGFβ also inhibited the expression of c-myc. Cotransfection with a Smad3 expressing plasmid further repressed rTERT transcription and c-myc expression, while cotransfection with the corresponding antisense Smad3 construct had the opposite effect. Mutation of an E-box in the rTERT promoter suppressed its activity, which could be further reduced by TGFβ treatment. In contrast, mutation at a Smad binding element enhanced promoter activity whose inhibition was impaired by TGFβ treatment. Thus TGFβ inhibition of rTERT gene expression was directly mediated by Smad3 via the Smad binding element, while c-myc appears to primarily regulate its constitutive or induced expression.

Rho proteins and cell-matrix interactions in cancer.

Dynamic interactions and dissolution of cell-extracellular matrix contacts are required steps to support cell growth and survival during cancer cell metastasis. Malignant cells acquire the ability to remodel extracellular matrix (ECM) and to modulate the expression of ECM receptors. Integrins are cellular receptors for molecules in the extracellular matrix. Integrin signaling is known to regulate metastatic cancer phenotypes by interacting synergistically with several signaling pathways, including the growth factor receptor pathway, the Ras-MAP kinase (Ras-MAPK) pathway and the Rho-effector pathway. In this mini-review, we discuss the functions of the Rho proteins and their relationship with other signaling pathways in matrix remodeling and integrin signaling of highly motile and invasive cancer cells.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including 13C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Macrophages Enhance Migration in Inflammatory Breast Cancer Cells via RhoC GTPase Signaling

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC’s extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC’s hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM “primes” the IBC cells’ cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins −6, −8, and −10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.