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302 Transcriptional Cooperation Between p53 and Estrogen Receptors in a Breast Cancer Model
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european journal of cancer 48, suppl. 5 (2012) S25–S288
301 Interleukin-6 Expression and JAK-STat Signalling in Prostate Cancer Stem Cells P. Kroon1 , P.A. Berry1 , D. Bhasin2 , C. Li2 , P.K. Li2 , M.J. Stower3 , V.M. Mann4 , M.S. Simms4 , N.J. Maitland1 , A.T. Collins1 . 1 University of York, Biology, York, United Kingdom, 2 Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, USA, 3 Department of Urology, York Hospital NHS Trust, York, United Kingdom, 4 Department of Urology, Castle Hill Hospital, Hull, United Kingdom Introduction: Prostate cancer is the most common cancer in men in the UK and is a major health problem worldwide. Currently there is no effective therapy for the recurring form of prostate cancer. Treatment failure may be due to the existence of cancer stem cells (CSCs). Gene expression profiling of prostate + cancer stem cells (CD44+ /a2 bhi 1 /CD133 ) showed that IL-6 and members of the JAK-STAT pathway were over-represented in CSCs relative to differentiated cells. If the JAK-STAT signalling pathway is indeed important for prostate CSC maintenance, this could represent a potential therapeutic target. Materials and Methods: The mRNA and protein expression of IL-6 in primary prostate cancer and benign cell cultures were analysed by qRT-PCR and ELISA. Immunofluorescence was performed to determine IL-6 receptor localization and Western blots were used to determine levels of phosphorylated STAT3. In vivo studies were carried out a panel of ‘near patient’ xenografts of prostate cancer in Rag2−/− g− C−/− mice, to determine the effect of inhibiting STAT3 directly on prostate cancer (stem) cell fate. Results and Discussion: IL-6 mRNA and protein were highly expressed in the CSC population compared to progenitor cells (CD133− /a2 blo 1 ) of primary prostate cell cultures. Significantly, this expression pattern was apparent in samples originating from patients with high Gleason grade tumours, but not from patients on hormone treatment or those with benign disease. The IL6 receptor was also expressed and STAT3 phosphorylated in both primary prostate cancer and benign samples indicating constitutive activation of the pathway. Inhibition of phosphorylated STAT3, in vivo, resulted in inhibition of tumour growth. In addition, a higher proportion of CD24+ luminal cells was observed. Conclusion: Prostate CSCs express high levels of IL-6 and the associated IL-6 receptor suggesting that these cells are dependent on the JAK-STAT signalling pathway via an autocrine feedback loop. Supporting evidence for this is that STAT3 is constitutively active in primary prostate cells. Preliminary in vivo data confirms this hypothesis, as treatment with a øSTAT3 inhibitor resulted in tumour growth inhibition. Initial results suggest that the basal tumour cells (CD44+ ) differentiate into a more luminal phenotype (CD24+ ) following inhibition of STAT3 phosphorylation, suggesting that the progenitor cells are dependent on the JAK-STAT signalling pathway to maintain an undifferentiated phenotype. 302 Transcriptional Cooperation Between p53 and Estrogen Receptors in a Breast Cancer Model M. Lion1 , A. Bisio1 , V. De Sanctis1 , Y. Ciribilli1 , T. Tebaldi2 , D. Menendez3 , M. Resnick3 , A. Inga1 . 1 University of Trento, Laboratory of Transcriptional Networks Centre for Integrative Biology (CIBIO), Trento, Italy, 2 University of Trento, Laboratory of Translational Genomics Centre for Integrative Biology (CIBIO), Trento, Italy, 3 National Institute of Environmental Health Sciences (NIEHS), Chromosome Stability Group Laboratory of Molecular Genetics, Research Triangle Park NC, USA Background: Previous reports have revealed a complex crosstalk between p53 and estrogen receptors (ERs) including transcriptional cooperation mediated by non-canonical cis-elements. The tumor suppressor p53 is a sequence-specific transcription factor activated by many stress signals that modulates genes involved mainly in apoptosis or cell cycle control. ERs are steroid hormone receptors and their primary response to estrogens is proliferation. Material and Methods: The breast cancer-derived MCF7 cells (p53 wild type; ERa and ERb − weakly − positive) were subjected to single or combination treatments with specific chemotherapeutic agents (doxorubicin or 5-fluorouracil) and the ER ligand 17b-estradiol (E2) to examine the impact of concomitant activation of p53 and ERs. Whole-genome expression analysis, followed up by quantitative real-time PCR (qPCR) and chromatin IP (ChIP) experiments were performed. Results: Our attention was focused on synergistic transcriptional cooperation between p53 and the ERs mediated through binding to canonical and/or noncanonical p53/estrogen receptor promoter response elements. A genome-wide transcriptome analysis was performed to identify genes nonresponsive or poorly responsive to p53 or ERs when acting alone but strongly regulated by their combined activation. We identified 201 genes as synergistically upregulated. Validation experiments were developed for 16 genes in MCF7 cells as well as in a MCF7 clone stably expressing shRNA to p53. qPCR was conducted in cells exposed or not to E2 and treated with genotoxic or nongenotoxic (nutlin-3a) p53 inducers. ChIP experiments were also performed to address direct p53 and ERs regulation of the selected genes. Some of these genes play an important role in inflammatory response, cell-cell
Sunday 8 − Tuesday 10 July 2012
communication, cell adhesion, development or differentiation (such as TLR5, KRT15, CDH26, NOTCH1, GDNF, INPP5D). The biological consequences of the identified p53-ER-dependent gene expression co-regulation are under investigation. Conclusions: p53 and ERs modulate distinct cellular responses but our results indicate that a synergistic cis-element-based transcriptional cooperation can also occur and appears to be related to non-canonical REs that are poorly or not responsive at all to p53 alone. Hence, the cooperation with ERs expands the p53 transcriptional network with implications for the appearance and possibly the treatments of cancer. 303 Mammaglobin Expression and Receptor Status in Primary and Recurrent Breast Cancer E.A. Baker1 , L. Hall2 , J. France2 , L. Noor1 , D. Whetter1 , P. Bhaskar1 . 1 University Hospital of North Tees, Professorial Unit of Surgery, Stockton on Tees, United Kingdom, 2 University Hospital of North Tees, Histopathology Department, Stockton on Tees, United Kingdom Background: Human mammaglobin has been reported to be exclusively expressed in mammary epithelium and over expressed in some breast cancers. The mammaglobin protein is dramatically increased in proliferating breast cells and its production ceases upon breast epithelial cell differentiation. Therefore, mammaglobin synthesis may be involved in breast cell proliferation which would correlate with the over expression seen in some breast cancers. Materials and Methods: 183 breast specimens were analysed by immunohistochemistry for mammaglobin A expression; 17 non-cancerous breast tissue, 143 were primary breast cancers and 23 were recurrent breast cancers. Stained sections were screened under the microscope with sections regarded as positive when >10% of lesional cells stained positive. For comparison purposes histological grade, tumour type, tumour size, ER, PR, Her-2 status and presence/absence of nodal metastasis were recorded. Controls were benign breast conditions. The study had ethics approval. Results: Positive mammaglobin expression was observed in 52% breast samples studied (52% primary and 48% recurrent). Positive expression was associated with benign or low grade tumours with 59% benign, 67% grade 1, 53% grade 2 and 41% grade 3 breast tumours demonstrating positive expression. Of the 21 patients with recurrent breast cancer, the same mammaglobin expression was observed in only 43% paired primary and recurrent tumour samples. Two of these patients had a second tumour recurrence but both of these samples had negative mammaglobin expression. Mammaglobin expression in primary tumours positively correlated with both ER status (58% correlation; p < 0.05 Chi-squared) and PR status (61% correlation). There was no correlation with lymphatic invasion, tumour size or HER-2 status. Conclusions: Positive mammaglobin protein expression was found in a greater proportion of benign and low grade breast tumours than higher grade. The expression in primary tumours was significantly associated with both ER and PR status. Since positive ER status and lower tumour grade are linked with a better prognosis for breast cancer patients, then mammaglobin A protein expression may also be associated with better prognosis. 304 Spontaneous Hybrids Between Breast Cancer and Multipotent Stromal Cells Acquire a Mixed Gene Expression Profile While Maintaining the Histopathological Appearance of Breast Carcinoma A. Lorico1 , J. Mercapide1 , G. Rappa1 . 1 Cancer Research Program, Roseman University of Health Sciences, Henderson, NV, USA Introduction: In the course of breast cancer progression, cell subpopulations exhibiting cytogenetic abnormalities along with invasive/prometastatic features are generated. Some of these cells break free from the primary tumor, invade the microvasculature, travel and establish foci at distant sites. Sequential genetic mutations, epithelial-mesenchymal transition, interaction with local stroma as well as formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report here the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSC) and two different breast carcinoma cell lines. Materials and Methods: We co-cultured human MDA-MB-231 (MDA) or MA11 breast carcinoma cells with MSC under conditions favoring the selection of hybrids. Hybrids were characterized in vitro, and implanted into immunedeficient mice. The histopathology, expression profile and coding SNPs of the xenografts were investigated. Results and Discussion: Hybrids showed a predominant mesenchymal morphology, mixed gene expression profiles and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphology, while maintaining a mixed breast
european journal of cancer 48, suppl. 5 (2012) S25–S288
301 Interleukin-6 Expression and JAK-STat Signalling in Prostate Cancer Stem Cells P. Kroon1 , P.A. Berry1 , D. Bhasin2 , C. Li2 , P.K. Li2 , M.J. Stower3 , V.M. Mann4 , M.S. Simms4 , N.J. Maitland1 , A.T. Collins1 . 1 University of York, Biology, York, United Kingdom, 2 Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy, The Ohio State University, Columbus, USA, 3 Department of Urology, York Hospital NHS Trust, York, United Kingdom, 4 Department of Urology, Castle Hill Hospital, Hull, United Kingdom Introduction: Prostate cancer is the most common cancer in men in the UK and is a major health problem worldwide. Currently there is no effective therapy for the recurring form of prostate cancer. Treatment failure may be due to the existence of cancer stem cells (CSCs). Gene expression profiling of prostate + cancer stem cells (CD44+ /a2 bhi 1 /CD133 ) showed that IL-6 and members of the JAK-STAT pathway were over-represented in CSCs relative to differentiated cells. If the JAK-STAT signalling pathway is indeed important for prostate CSC maintenance, this could represent a potential therapeutic target. Materials and Methods: The mRNA and protein expression of IL-6 in primary prostate cancer and benign cell cultures were analysed by qRT-PCR and ELISA. Immunofluorescence was performed to determine IL-6 receptor localization and Western blots were used to determine levels of phosphorylated STAT3. In vivo studies were carried out a panel of ‘near patient’ xenografts of prostate cancer in Rag2−/− g− C−/− mice, to determine the effect of inhibiting STAT3 directly on prostate cancer (stem) cell fate. Results and Discussion: IL-6 mRNA and protein were highly expressed in the CSC population compared to progenitor cells (CD133− /a2 blo 1 ) of primary prostate cell cultures. Significantly, this expression pattern was apparent in samples originating from patients with high Gleason grade tumours, but not from patients on hormone treatment or those with benign disease. The IL6 receptor was also expressed and STAT3 phosphorylated in both primary prostate cancer and benign samples indicating constitutive activation of the pathway. Inhibition of phosphorylated STAT3, in vivo, resulted in inhibition of tumour growth. In addition, a higher proportion of CD24+ luminal cells was observed. Conclusion: Prostate CSCs express high levels of IL-6 and the associated IL-6 receptor suggesting that these cells are dependent on the JAK-STAT signalling pathway via an autocrine feedback loop. Supporting evidence for this is that STAT3 is constitutively active in primary prostate cells. Preliminary in vivo data confirms this hypothesis, as treatment with a øSTAT3 inhibitor resulted in tumour growth inhibition. Initial results suggest that the basal tumour cells (CD44+ ) differentiate into a more luminal phenotype (CD24+ ) following inhibition of STAT3 phosphorylation, suggesting that the progenitor cells are dependent on the JAK-STAT signalling pathway to maintain an undifferentiated phenotype. 302 Transcriptional Cooperation Between p53 and Estrogen Receptors in a Breast Cancer Model M. Lion1 , A. Bisio1 , V. De Sanctis1 , Y. Ciribilli1 , T. Tebaldi2 , D. Menendez3 , M. Resnick3 , A. Inga1 . 1 University of Trento, Laboratory of Transcriptional Networks Centre for Integrative Biology (CIBIO), Trento, Italy, 2 University of Trento, Laboratory of Translational Genomics Centre for Integrative Biology (CIBIO), Trento, Italy, 3 National Institute of Environmental Health Sciences (NIEHS), Chromosome Stability Group Laboratory of Molecular Genetics, Research Triangle Park NC, USA Background: Previous reports have revealed a complex crosstalk between p53 and estrogen receptors (ERs) including transcriptional cooperation mediated by non-canonical cis-elements. The tumor suppressor p53 is a sequence-specific transcription factor activated by many stress signals that modulates genes involved mainly in apoptosis or cell cycle control. ERs are steroid hormone receptors and their primary response to estrogens is proliferation. Material and Methods: The breast cancer-derived MCF7 cells (p53 wild type; ERa and ERb − weakly − positive) were subjected to single or combination treatments with specific chemotherapeutic agents (doxorubicin or 5-fluorouracil) and the ER ligand 17b-estradiol (E2) to examine the impact of concomitant activation of p53 and ERs. Whole-genome expression analysis, followed up by quantitative real-time PCR (qPCR) and chromatin IP (ChIP) experiments were performed. Results: Our attention was focused on synergistic transcriptional cooperation between p53 and the ERs mediated through binding to canonical and/or noncanonical p53/estrogen receptor promoter response elements. A genome-wide transcriptome analysis was performed to identify genes nonresponsive or poorly responsive to p53 or ERs when acting alone but strongly regulated by their combined activation. We identified 201 genes as synergistically upregulated. Validation experiments were developed for 16 genes in MCF7 cells as well as in a MCF7 clone stably expressing shRNA to p53. qPCR was conducted in cells exposed or not to E2 and treated with genotoxic or nongenotoxic (nutlin-3a) p53 inducers. ChIP experiments were also performed to address direct p53 and ERs regulation of the selected genes. Some of these genes play an important role in inflammatory response, cell-cell
Sunday 8 − Tuesday 10 July 2012
communication, cell adhesion, development or differentiation (such as TLR5, KRT15, CDH26, NOTCH1, GDNF, INPP5D). The biological consequences of the identified p53-ER-dependent gene expression co-regulation are under investigation. Conclusions: p53 and ERs modulate distinct cellular responses but our results indicate that a synergistic cis-element-based transcriptional cooperation can also occur and appears to be related to non-canonical REs that are poorly or not responsive at all to p53 alone. Hence, the cooperation with ERs expands the p53 transcriptional network with implications for the appearance and possibly the treatments of cancer. 303 Mammaglobin Expression and Receptor Status in Primary and Recurrent Breast Cancer E.A. Baker1 , L. Hall2 , J. France2 , L. Noor1 , D. Whetter1 , P. Bhaskar1 . 1 University Hospital of North Tees, Professorial Unit of Surgery, Stockton on Tees, United Kingdom, 2 University Hospital of North Tees, Histopathology Department, Stockton on Tees, United Kingdom Background: Human mammaglobin has been reported to be exclusively expressed in mammary epithelium and over expressed in some breast cancers. The mammaglobin protein is dramatically increased in proliferating breast cells and its production ceases upon breast epithelial cell differentiation. Therefore, mammaglobin synthesis may be involved in breast cell proliferation which would correlate with the over expression seen in some breast cancers. Materials and Methods: 183 breast specimens were analysed by immunohistochemistry for mammaglobin A expression; 17 non-cancerous breast tissue, 143 were primary breast cancers and 23 were recurrent breast cancers. Stained sections were screened under the microscope with sections regarded as positive when >10% of lesional cells stained positive. For comparison purposes histological grade, tumour type, tumour size, ER, PR, Her-2 status and presence/absence of nodal metastasis were recorded. Controls were benign breast conditions. The study had ethics approval. Results: Positive mammaglobin expression was observed in 52% breast samples studied (52% primary and 48% recurrent). Positive expression was associated with benign or low grade tumours with 59% benign, 67% grade 1, 53% grade 2 and 41% grade 3 breast tumours demonstrating positive expression. Of the 21 patients with recurrent breast cancer, the same mammaglobin expression was observed in only 43% paired primary and recurrent tumour samples. Two of these patients had a second tumour recurrence but both of these samples had negative mammaglobin expression. Mammaglobin expression in primary tumours positively correlated with both ER status (58% correlation; p < 0.05 Chi-squared) and PR status (61% correlation). There was no correlation with lymphatic invasion, tumour size or HER-2 status. Conclusions: Positive mammaglobin protein expression was found in a greater proportion of benign and low grade breast tumours than higher grade. The expression in primary tumours was significantly associated with both ER and PR status. Since positive ER status and lower tumour grade are linked with a better prognosis for breast cancer patients, then mammaglobin A protein expression may also be associated with better prognosis. 304 Spontaneous Hybrids Between Breast Cancer and Multipotent Stromal Cells Acquire a Mixed Gene Expression Profile While Maintaining the Histopathological Appearance of Breast Carcinoma A. Lorico1 , J. Mercapide1 , G. Rappa1 . 1 Cancer Research Program, Roseman University of Health Sciences, Henderson, NV, USA Introduction: In the course of breast cancer progression, cell subpopulations exhibiting cytogenetic abnormalities along with invasive/prometastatic features are generated. Some of these cells break free from the primary tumor, invade the microvasculature, travel and establish foci at distant sites. Sequential genetic mutations, epithelial-mesenchymal transition, interaction with local stroma as well as formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report here the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSC) and two different breast carcinoma cell lines. Materials and Methods: We co-cultured human MDA-MB-231 (MDA) or MA11 breast carcinoma cells with MSC under conditions favoring the selection of hybrids. Hybrids were characterized in vitro, and implanted into immunedeficient mice. The histopathology, expression profile and coding SNPs of the xenografts were investigated. Results and Discussion: Hybrids showed a predominant mesenchymal morphology, mixed gene expression profiles and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphology, while maintaining a mixed breast