Morwenna Muir

WWOX gene expression abolishes ovarian cancer tumorigenicity in vivo and decreases attachment to fibronectin via integrin alpha3.

The WW domain-containing oxidoreductase (WWOX) gene is located at FRA16D, a common fragile site involved in human cancer. Targeted deletion of Wwox in mice causes increased spontaneous tumor incidence, confirming that WWOX is a bona fide tumor suppressor gene. We show that stable transfection of WWOX into human PEO1 ovarian cancer cells, containing homozygous WWOX deletion, abolishes in vivo tumorigenicity, but this does not correlate with alteration of in vitro growth. Rather, WWOX restoration in PEO1, or WWOX overexpression in SKOV3 ovarian cancer cells, results in reduced attachment and migration on fibronectin, an extracellular matrix component linked to peritoneal metastasis. Conversely, siRNA-mediated knockdown of endogenous WWOX in A2780 ovarian cancer cells increases adhesion to fibronectin. In addition, whereas there is no WWOX-dependent difference in cell death in adherent cells, WWOX-transfected cells in suspension culture display a proapoptotic phenotype. We further show that WWOX expression reduces membranous integrin alpha(3) protein but not integrin alpha(3) mRNA levels, and that adhesion of PEO1 cells to fibronectin is predominantly mediated through integrin alpha(3). We therefore propose that WWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix and by inducing apoptosis in detached cells. Consistent with this, the suppression of PEO1 tumorigenicity by WWOX can be partially overcome by implanting these tumor cells in Matrigel. These data suggest a possible role for the loss of WWOX in the peritoneal dissemination of human ovarian cancer cells.

Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.

Summary Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8+ T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8+ T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK’s immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8+ T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

Gonadotropin-Releasing Hormone Receptor Levels and Cell Context Affect Tumor Cell Responses to Agonist In vitro and In vivo

Activation of gonadotropin-releasing hormone (GnRH) receptors inhibits proliferation of transformed cells derived from reproductive tissues and in transfected cell lines. Hence, GnRH receptors represent a therapeutic target for direct action of GnRH analogues on certain proliferating cells. However, more cell biological data are required to develop this particular application of GnRH analogues. Therefore, we compared the effects of GnRH receptor activation in transfected HEK293 cells (HEK293([SCL60])) with transfected human ovarian cancer cell lines SKOV3 and EFO21, human hepatoblastoma HepG2 cells, and rat neuroblastoma B35 cells. Marked differences in receptor levels, magnitude of inositol phosphate generation, and dynamics of inositol phosphate turnover occurred in the different cells. Activation of GnRH receptors, expressed at high or moderate levels, inhibited the growth of HEK293([SCL60]) and B35 cells, respectively. Western blotting detected markers of apoptosis [cleaved poly(ADP-ribose) polymerase, caspase-9] in HEK293([SCL60]) and B35 following treatment with 100 nmol/L d-Trp(6)-GnRH-I. Cell growth inhibition was partially or completely rescued with inhibitor Q-VD-OPh or Ro32-0432. Low levels of GnRH receptor expression in transfected SKOV3, EFO21, or HepG2 activated intracellular signaling but did not induce apoptosis or significantly affect cell proliferation. Tumor xenografts prepared from HEK293([SCL60]) regressed during treatment with d-Trp(6)-GnRH-I and growth of xenografts derived from transfected B35 was slowed. SKOV3 xenografts were not growth inhibited. Therefore, differences in levels of GnRH receptor and signaling differentially affect the apoptotic machinery within cell lines and contribute to the cell type-specific effects of GnRH on growth. Further studies should exploit the growth-inhibitory potential of GnRH receptor activation in abnormal cells in diseased human tissues.

Trastuzumab and Pertuzumab Produce Changes in Morphology and Estrogen Receptor Signaling in Ovarian Cancer Xenografts Revealing New Treatment Strategies

Purpose: The aim of this study was to investigate the antitumor effects of HER2-directed combination therapy in ovarian cancer xenograft models to evaluate their potential. The combinations of trastuzumab and pertuzumab, and trastuzumab and aromatase inhibitor therapy were investigated. Experimental Design: The effects of trastuzumab, pertuzumab, and letrozole on growth response, apoptosis, morphology, and gene and protein expression were evaluated in the SKOV3 ovarian cancer cell line xenograft and a panel of five human ovarian xenografts derived directly from clinical specimens. Results: The combination of HER2-directed antibodies showed enhanced antitumor activity compared with single antibody therapy in the SKOV3 xenograft model. Apoptosis, morphology, and estrogen-regulated gene expression were modulated by these antibodies in both spatial and temporal manners. A panel of ovarian cancer xenografts showed differential growth responses to the combination of trastuzumab and pertuzumab. High HER2 expression and increasing HER3 protein expression on treatment were associated with growth response. In trastuzumab-treated SKOV3 tumors, there was a change in tumor morphology, with a reduction in frequency of estrogen receptor alpha (ERα)-negative clear cell areas. Trastuzumab, but not pertuzumab, increased expression of ERα in SKOV3 xenografts when analyzed by quantitative immunofluorescence. ERα and downstream signaling targets were modulated by trastuzumab alone and in combination. Trastuzumab enhanced the responsiveness of SKOV3 xenografts to letrozole when given in combination. Conclusions: These data suggest that trastuzumab in combination with pertuzumab could be an effective approach in high HER2-expressing ovarian cancers and could also enhance sensitivity to endocrine therapy in ERα-positive ovarian cancer. Clin Cancer Res; 17(13); 4451–61. ©2011 AACR.

Dynamic changes in gene expression in vivo predict prognosis of tamoxifen-treated patients with breast cancer

IntroductionTamoxifen is the most widely prescribed anti-estrogen treatment for patients with estrogen receptor (ER)-positive breast cancer. However, there is still a need for biomarkers that reliably predict endocrine sensitivity in breast cancers and these may well be expressed in a dynamic manner.MethodsIn this study we assessed gene expression changes at multiple time points (days 1, 2, 4, 7, 14) after tamoxifen treatment in the ER-positive ZR-75-1 xenograft model that displays significant changes in apoptosis, proliferation and angiogenesis within 2 days of therapy.ResultsHierarchical clustering identified six time-related gene expression patterns, which separated into three groups: two with early/transient responses, two with continuous/late responses and two with variable response patterns. The early/transient response represented reductions in many genes that are involved in cell cycle and proliferation (e.g. BUB1B, CCNA2, CDKN3, MKI67, UBE2C), whereas the continuous/late changed genes represented the more classical estrogen response genes (e.g. TFF1, TFF3, IGFBP5). Genes and the proteins they encode were confirmed to have similar temporal patterns of expression in vitro and in vivo and correlated with reduction in tumour volume in primary breast cancer. The profiles of genes that were most differentially expressed on days 2, 4 and 7 following treatment were able to predict prognosis, whereas those most changed on days 1 and 14 were not, in four tamoxifen treated datasets representing a total of 404 patients.ConclusionsBoth early/transient/proliferation response genes and continuous/late/estrogen-response genes are able to predict prognosis of primary breast tumours in a dynamic manner. Temporal expression of therapy-response genes is clearly an important factor in characterising the response to endocrine therapy in breast tumours which has significant implications for the timing of biopsies in neoadjuvant biomarker studies.

Defining the molecular response to trastuzumab, pertuzumab and combination therapy in ovarian cancer

Background:Trastuzumab and pertuzumab target the Human Epidermal growth factor Receptor 2 (HER2). Combination therapy has been shown to provide enhanced antitumour activity; however, the downstream signalling to explain how these drugs mediate their response is not clearly understood.Methods:Transcriptome profiling was performed after 4 days of trastuzumab, pertuzumab and combination treatment in human ovarian cancer in vivo. Signalling pathways identified were validated and investigated in primary ovarian xenografts at the protein level and across a timeseries.Results:A greater number and variety of genes were differentially expressed by the combination of antibody therapies compared with either treatment alone. Protein levels of cyclin-dependent kinase inhibitors p21 and p27 were increased in response to both agents and further by the combination; pERK signalling was inhibited by all treatments; but only pertuzumab inhibited pAkt signalling. The expression of proliferation, apoptosis, cell division and cell-cycle markers was distinct in a panel of primary ovarian cancer xenografts, suggesting the heterogeneity of response in ovarian cancer and a need to establish predictive biomarkers.Conclusion:This first comprehensive study of the molecular response to trastuzumab, pertuzumab and combined therapy in vivo highlights both common and distinct downstream effects to agents used alone or in combination, suggesting that complementary pathways may be involved.

Stable XIAP knockdown clones of HCT116 colon cancer cells are more sensitive to TRAIL, taxanes and irradiation in vitro

PurposeTo develop a model of X-linked inhibitor of apoptosis (XIAP) down regulation in colorectal cancer cell lines. This may be used to determine whether combination strategies have clinical potential.MethodsA series of clones were developed using short hairpin RNA (shRNA) against XIAP stably expressed in HCT116 cells. XIAP mRNA and protein levels were established by RT-PCR and Immunoblot, respectively. GeneChip microarrays confirmed XIAP knockdown and absence of compensation by other IAP members.ResultsFour XIAP knockdown cell lines show 82–93% reduction in XIAP mRNA and 67–89% reduction in protein when compared to four luciferase control cell lines. XIAP knockdown sensitises cells to rhTRAIL by a factor of 3, to paclitaxel and docetaxel by a factor of >2 and, to a lesser extent, radiotherapy (20% enhancement).ConclusionsClinical trials with XIAP antisense continue, and these data suggest combination studies with agents such as rhTRAIL and taxanes should be undertaken.

Focal adhesion kinase is required for β-catenin-induced mobilization of epidermal stem cells

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates signals downstream of integrin and growth factor activation. Previously, we have shown that skin-specific loss of fak prevents chemically induced skin carcinogenesis in mice following phorbol ester treatment. In this study, we show that skin-specific deletion of fak prevents mobilization of stem cells within the bulge region of the hair follicle, which are the precursors of papillomas following phorbol ester treatment. We also show that phorbol ester treatment results in activation of-catenin within the skin and that FAK is required for β-catenin-induced stem cell mobilization. In addition, inhibition of Src kinase activity, a major binding partner of FAK also prevents stem cell mobilization. We show that FAK is required for the nuclear localization of β-catenin in the skin following phorbol ester treatment and the transcriptional activation of the β-catenin target gene c-Myc. This provides the first evidence of cross-talk between integrin and Wnt signalling pathways in the control of epidermal stem cells and the early events associated with skin carcinogenesis.

Diversity of matriptase expression level and function in breast cancer

Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent.

Selective tyrosine kinase inhibition of insulin-like growth factor-1 receptor inhibits human and mouse breast cancer-induced bone cell activity, bone remodeling, and osteolysis.

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer.