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Tamoxifen Resistance occurs through SIAH2 in Estrogen Receptor Positive Breast Cancer
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
CD8 T cell, B cell and regulatory T cell infiltration when compared to mice without tumor. Lymphocyte infiltration was further characterized by immunohistochemistry (IHC) staining of the DLN. Additionally, the interactions between fluorescently-labeled T cells and tumor cells in the metastatic lymph node and primary tumor were visualized by intravital 2-photon laser-scanning microscopy on tumorbearing mice at days 2-10 after tumor inoculation. Results: Three days after tumor challenge, the lymphocyte distribution in the DLN showed increased B cell infiltration and a drop in CD4 T cells compared to unchallenged mice in all tumor cell lines (Figure). At day 10, there was a shift in percent cellularity among lymphocytes in the DLN of CCL3 and CCL4-secreting tumors but not in WT tumors. The greatest change was an increase percent CD8 T cells in the DLN of mice injected with CCL3-secreting tumors. In vivo imaging of the DLN of tumor-bearing mice at day 3 showed subcapsular metastasis of tumor in the B cell follicle of the DLN in all tumor cell lines (also confirmed by IHC) and decreased motility of T cells around CCL4-secreting metastatic tumor cells. Conclusions: Mice challenged with CT26 colon cancer cells that secrete chemokine CCL3 are preferentially protected from tumor development compared to mice injected with WT or CCL4-secreting cells due to the noted differential recruitment of B cells and CD8 T cells in the draining lymph nodes by the chemokine CCL3. This variation of recruitment is likely due to the interaction of CCL3 with other receptors besides CCR5, such as CCR1 and CCR3, whereas CCL4 only attracts immune cells via the CCR5 receptor.
play a role in tamoxifen resistance via Seven in Absentia Homolog 2 (SIAH2), an E3 ubiquitin protein ligase. SIAH2 has been shown to regulate HIF-1a stability via regulation of protein stability of prolyl hydroxylases. Here we assess whether SIAH2 modulates HIF-1a expression in ER positive breast cancers, and whether SIAH2 modulates ER function in response to tamoxifen treatment. Methods: Two human breast cancer cell lines obtained from ATCC were included in this study – MCF-7 and MDA-MB-231, which are ER positive and negative cell lines, respectively. SIAH2 and ERa were selectively knocked down with small-interfering RNA. Knockdowns were confirmed with Western Blots and quantitative real-time polymerase chain reaction (qRT-PCR) was also used to assess mRNA. Knockdowns of both proteins were then treated with tamoxifen at increasing doses. Lysate and RNA extraction was then assessed to evaluate the dose response. MCF-7 was also treated with tamoxifen and bestradiol at increasing time points to assess changes in protein expression of SIAH2 and ER-a. Results: A bioinformatic analysis revealed that SIAH2 expression is strongly correlated with ER positivity and the SIAH2 gene bears two ER binding sites. In vitro experiments showed that selective knockdown of SIAH2 resulted in down-regulation of ER-a, as well as HIF1a by western blot and qRT-PCR, and knockdown of ER-a caused a significant down-regulation of HIF1a. Treatment of MCF-7 cell line with tamoxifen at increasing time points increased SIAH2 up to 8 hours, whereas ERa increased up to 24 hours. Treatment of MCF-7 with b-estradiol showed a decrease in ER-a, as well as induction of HIF-1a by western blot. Conclusions: Previous work has outlined the importance of SIAH2 in the treatment of ER-positive breast cancer with anti-estrogen therapies. SIAH2 likely functions through direct interactions with ER-a, and indirectly with HIF-1a, as a tumor suppressor in the hypoxic setting of advanced tumors. This process appears to be a time-dependent fashion, and may be critical for the development of resistance to this drug. Further experiments will focus on grasping a better understanding of the critical role of SIAH2 in tamoxifen resistance.
FIG. Changes in lymphocyte % cellularity within the draining lymph node (DLN) of mice inoculated with WT, CCL3- or CCL4secreting CT26 tumor. Under normal conditions, CD4 T cells contributes more to the lymph node cellularity than other lymphocytes. When challenged with tumors, within 3 days, B cells contribution is greater in the DLN and remains greater over the 10 day period in all mice except those injected with the CCL3-secreting tumor. CCL3 tumors have the greatest increase in CD8 T cell recruitment in the DLN at day 10.
17.6. Tamoxifen Resistance occurs through SIAH2 in Estrogen Receptor Positive Breast Cancer. R. B. Interiano,1,2 J. Yang,1 A. M. Davidoff1,2; 1St. Jude Children’s Research Hospital, Memphis, TN, USA; 2University Of Tennessee Health Science Center - Department Of Surgery, Memphis, TN, USA Introduction: Breast cancer is the most common malignancy in women worldwide and is frequently treated with tamoxifen, a selective estrogen receptor (ER) modulator. Resistance to tamoxifen has however poised a difficult dilemma in estrogen receptor positive breast cancer. The pathways to this resistance are incompletely understood but may be related to tumor hypoxia. Interestingly, our preliminary data indicate that hypoxia inducible factor 1-a (HIF-1a) may autonomously
FIG. 1. Bio-informatics analysis shows SIAH2 gene has two estrogen receptor binding sites. (p<10 6). The correlation of gene expression of SIAH2 and other genes from TCGA breast cancer data was analyzed by Regulome program. The number represents 22 autosomes and X/Y chromosome. The well-known ER target gene with genomiclocation was shown in the circus.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
FIG. 2. MCF-7 cells were plated with siRNA, knockdown of SIAH2 and ERa, and harvested after 72 hours of incubation. SIAH2 knockdown with the siSIAH 2#1 showed decrease in SIAH2, with subsequent decrease in HIF-1a MDA-MB-231 was then also treated similarly and western blot analysis performed.
FIG. 3. MCF-7 cells were plated in DMEM, after 48 hours of incubation, cells were then hormone starved for 72 hours. Subsequently, the cells were then treated with increasing doses of tamoxifen or b-estradiol for 24 hours. Western blot analysis was then performed.
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FIG. 5. MCF-7 cells were treated as in Figure 2, with subsequent RNA extraction at time of harvest. The extract was then analyzed via qRT-PCR to assess VEGF, SIAH2, and ERa.
FIG. 6. MCF-7 cells were treated with tamoxifen and b-estradiol after hormone starvation in charcoal-stripped DMEM for increasing amount of time. Cells were then harvested, and western blot performed.
17.7. Tissue-Restricted Autoimmunity Leads to the Development of Lung Allograft Rejection. A. Bharat,1 V. Subramanian,3 M. Decamp,1 D. Kriesel,2 A. Gelman,2 T. Mohanakumar2; 1Northwestern University - Thoracic/ General Surgery/Feinberg School Of Medicine, Chicago, IL, USA; 2Washington University - Pathology And Immunology/ Surgery, St. Louis, MO, USA; 3University Of Mississippi General Surgery, Jackson, Mississippi, USA
FIG. 4. MCF-7 cells were plated with siRNA knockdown of SIAH2 and ERa After 72 hours of incubation, RNA extraction was performed with subsequent qRT-PCR analysis of the mRNA to assess SIAH,ERa, and HIF-1a.
Introduction: Recent data shows that a large number of lung transplant (LTx) recipients develop de novo antibodies (Abs) against lung-restricted autoantigens, namely k-alpha 1 tubulin (KAT) and collagen type V (colV). The role of these auto-Abs in allograft rejection remains unclear. Using the murine model of lung transplant we tested the hypothesis that auto-Abs can abrogate immunosuppression-mediated tolerance of lung allografts. Further, to avoid the
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
CD8 T cell, B cell and regulatory T cell infiltration when compared to mice without tumor. Lymphocyte infiltration was further characterized by immunohistochemistry (IHC) staining of the DLN. Additionally, the interactions between fluorescently-labeled T cells and tumor cells in the metastatic lymph node and primary tumor were visualized by intravital 2-photon laser-scanning microscopy on tumorbearing mice at days 2-10 after tumor inoculation. Results: Three days after tumor challenge, the lymphocyte distribution in the DLN showed increased B cell infiltration and a drop in CD4 T cells compared to unchallenged mice in all tumor cell lines (Figure). At day 10, there was a shift in percent cellularity among lymphocytes in the DLN of CCL3 and CCL4-secreting tumors but not in WT tumors. The greatest change was an increase percent CD8 T cells in the DLN of mice injected with CCL3-secreting tumors. In vivo imaging of the DLN of tumor-bearing mice at day 3 showed subcapsular metastasis of tumor in the B cell follicle of the DLN in all tumor cell lines (also confirmed by IHC) and decreased motility of T cells around CCL4-secreting metastatic tumor cells. Conclusions: Mice challenged with CT26 colon cancer cells that secrete chemokine CCL3 are preferentially protected from tumor development compared to mice injected with WT or CCL4-secreting cells due to the noted differential recruitment of B cells and CD8 T cells in the draining lymph nodes by the chemokine CCL3. This variation of recruitment is likely due to the interaction of CCL3 with other receptors besides CCR5, such as CCR1 and CCR3, whereas CCL4 only attracts immune cells via the CCR5 receptor.
play a role in tamoxifen resistance via Seven in Absentia Homolog 2 (SIAH2), an E3 ubiquitin protein ligase. SIAH2 has been shown to regulate HIF-1a stability via regulation of protein stability of prolyl hydroxylases. Here we assess whether SIAH2 modulates HIF-1a expression in ER positive breast cancers, and whether SIAH2 modulates ER function in response to tamoxifen treatment. Methods: Two human breast cancer cell lines obtained from ATCC were included in this study – MCF-7 and MDA-MB-231, which are ER positive and negative cell lines, respectively. SIAH2 and ERa were selectively knocked down with small-interfering RNA. Knockdowns were confirmed with Western Blots and quantitative real-time polymerase chain reaction (qRT-PCR) was also used to assess mRNA. Knockdowns of both proteins were then treated with tamoxifen at increasing doses. Lysate and RNA extraction was then assessed to evaluate the dose response. MCF-7 was also treated with tamoxifen and bestradiol at increasing time points to assess changes in protein expression of SIAH2 and ER-a. Results: A bioinformatic analysis revealed that SIAH2 expression is strongly correlated with ER positivity and the SIAH2 gene bears two ER binding sites. In vitro experiments showed that selective knockdown of SIAH2 resulted in down-regulation of ER-a, as well as HIF1a by western blot and qRT-PCR, and knockdown of ER-a caused a significant down-regulation of HIF1a. Treatment of MCF-7 cell line with tamoxifen at increasing time points increased SIAH2 up to 8 hours, whereas ERa increased up to 24 hours. Treatment of MCF-7 with b-estradiol showed a decrease in ER-a, as well as induction of HIF-1a by western blot. Conclusions: Previous work has outlined the importance of SIAH2 in the treatment of ER-positive breast cancer with anti-estrogen therapies. SIAH2 likely functions through direct interactions with ER-a, and indirectly with HIF-1a, as a tumor suppressor in the hypoxic setting of advanced tumors. This process appears to be a time-dependent fashion, and may be critical for the development of resistance to this drug. Further experiments will focus on grasping a better understanding of the critical role of SIAH2 in tamoxifen resistance.
FIG. Changes in lymphocyte % cellularity within the draining lymph node (DLN) of mice inoculated with WT, CCL3- or CCL4secreting CT26 tumor. Under normal conditions, CD4 T cells contributes more to the lymph node cellularity than other lymphocytes. When challenged with tumors, within 3 days, B cells contribution is greater in the DLN and remains greater over the 10 day period in all mice except those injected with the CCL3-secreting tumor. CCL3 tumors have the greatest increase in CD8 T cell recruitment in the DLN at day 10.
17.6. Tamoxifen Resistance occurs through SIAH2 in Estrogen Receptor Positive Breast Cancer. R. B. Interiano,1,2 J. Yang,1 A. M. Davidoff1,2; 1St. Jude Children’s Research Hospital, Memphis, TN, USA; 2University Of Tennessee Health Science Center - Department Of Surgery, Memphis, TN, USA Introduction: Breast cancer is the most common malignancy in women worldwide and is frequently treated with tamoxifen, a selective estrogen receptor (ER) modulator. Resistance to tamoxifen has however poised a difficult dilemma in estrogen receptor positive breast cancer. The pathways to this resistance are incompletely understood but may be related to tumor hypoxia. Interestingly, our preliminary data indicate that hypoxia inducible factor 1-a (HIF-1a) may autonomously
FIG. 1. Bio-informatics analysis shows SIAH2 gene has two estrogen receptor binding sites. (p<10 6). The correlation of gene expression of SIAH2 and other genes from TCGA breast cancer data was analyzed by Regulome program. The number represents 22 autosomes and X/Y chromosome. The well-known ER target gene with genomiclocation was shown in the circus.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
FIG. 2. MCF-7 cells were plated with siRNA, knockdown of SIAH2 and ERa, and harvested after 72 hours of incubation. SIAH2 knockdown with the siSIAH 2#1 showed decrease in SIAH2, with subsequent decrease in HIF-1a MDA-MB-231 was then also treated similarly and western blot analysis performed.
FIG. 3. MCF-7 cells were plated in DMEM, after 48 hours of incubation, cells were then hormone starved for 72 hours. Subsequently, the cells were then treated with increasing doses of tamoxifen or b-estradiol for 24 hours. Western blot analysis was then performed.
517
FIG. 5. MCF-7 cells were treated as in Figure 2, with subsequent RNA extraction at time of harvest. The extract was then analyzed via qRT-PCR to assess VEGF, SIAH2, and ERa.
FIG. 6. MCF-7 cells were treated with tamoxifen and b-estradiol after hormone starvation in charcoal-stripped DMEM for increasing amount of time. Cells were then harvested, and western blot performed.
17.7. Tissue-Restricted Autoimmunity Leads to the Development of Lung Allograft Rejection. A. Bharat,1 V. Subramanian,3 M. Decamp,1 D. Kriesel,2 A. Gelman,2 T. Mohanakumar2; 1Northwestern University - Thoracic/ General Surgery/Feinberg School Of Medicine, Chicago, IL, USA; 2Washington University - Pathology And Immunology/ Surgery, St. Louis, MO, USA; 3University Of Mississippi General Surgery, Jackson, Mississippi, USA
FIG. 4. MCF-7 cells were plated with siRNA knockdown of SIAH2 and ERa After 72 hours of incubation, RNA extraction was performed with subsequent qRT-PCR analysis of the mRNA to assess SIAH,ERa, and HIF-1a.
Introduction: Recent data shows that a large number of lung transplant (LTx) recipients develop de novo antibodies (Abs) against lung-restricted autoantigens, namely k-alpha 1 tubulin (KAT) and collagen type V (colV). The role of these auto-Abs in allograft rejection remains unclear. Using the murine model of lung transplant we tested the hypothesis that auto-Abs can abrogate immunosuppression-mediated tolerance of lung allografts. Further, to avoid the