Yahya Elshimali - Academia.edu (original) (raw)
Papers by Yahya Elshimali
The Internet Journal of Pathology, 2009
International Journal of Clinical Investigation and Case Reports
Skene’s glands are a pair of female paraurethral glands with similar embryologic, physiologic, an... more Skene’s glands are a pair of female paraurethral glands with similar embryologic, physiologic, and pathologic features to the male prostate. Cysts and abscesses represent the most common pathological changes affecting Skene’s gland, whereas adenofibromas represent an extremely rare entity. Herein, we present a rare case report of adenofibroma in the Skene’s gland of a postmenopausal female.
One major impediment to the success of chemotherapy in cancer patients is the risk of developing ... more One major impediment to the success of chemotherapy in cancer patients is the risk of developing drug resistance. One mechanism leading to multidrug resistance in cancer therapy is through the ABC transporters. The eukaryotic ABC genes are organized as transporters containing two transmembrane and two ATP binding domains. Alternative splicing is one major mechanism in which a single gene generates functional diversity by producing different spliced isoforms. To determine the variability of ABC transporters, we performed exhaustive search of the NCBI database for ABC transporter genes, in addition to using the Alternative Splicing Annotation Project (ASAP) and the Alternative Splicing Database (ASD). We conducted immunostaining for MRP1 and MDR1 on tissue micro array of 23 breast cancer patients and RNA mediated annealing, selection, extension and ligation (RASL) assay for analysis of mRNA splicing isoforms which was performed on exon/exon target. Tumor cells from chemotherapy resistant patients show specific expression patterns of drug-resistant ATP-binding cassette transporter. Our data showed alternative splicing in 3 and 4 exon/exon borders in MDR1 and PGP genes. Two splicing events in MDR1 and one in PGP transcripts were predicted to be alternatively spliced in ASAP database. Interestingly, exon11/12 border in MDR1 is found to be alternatively spliced. In summary, protein microarray analysis demonstrated significant correlation between MRP1 expression and survival rate within 5 years. In contrast, MDRI protein expression did not correlate with 5 year survival rate. This may be due to variations in the MDR1 isoforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3531.
Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. ... more Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. MDA-MB-231 cells were transfected with WT-SMAD4, HA-ubiquitin, and the indicated shRNAs and incubated with BSA or PA in the presence or absence of 3 ng/ml TGF-beta1 for 2 h. The effective knockdown of USP9x was confirmed by Western blot analysis before ubiquitination assays. Nuclear SMAD4 monoubiquitination (Mono-Ub) was detected by anti-SMAD4 immunoprecipitation and immunoblot with HA-ubiquitin. The blot is a representative of 3 blots from 3 independent experiments. *P<0.05 vs Control (shControl/-TGF-beta/-PA); #P<0.01 vs shControl/-PA; $P<0.05 shControl/+PA.
Figure S3 FFA promotes TGF-beta-induced migration by activating ERK and USP9x, Related to Figure ... more Figure S3 FFA promotes TGF-beta-induced migration by activating ERK and USP9x, Related to Figure 3. A, BT549 cells were plated in Ibidi culture insert dishes (Madison, WI). Cells were cultured in complete medium to 80% confluence, inserts were removed to create a gap and cells were then incubated with BSA, PA, PA plus AZD6244 or WP1130 in the absence or presence of TGF-beta1. Migrating cells were photographed under a phase contrast microscope. B, The percentage of the wound closed was quantified from three independent replicates and is expressed as mean {plus minus} SD. *p<0.05 vs BSA/TGF-beta; #p<0.05 vs PA/TGF-beta.
Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented... more Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. B, *p<0.01 vs BSA; #p<0.01 PA+AZD vs PA. E, *p<0.01 vs EV/-PA; #p<0.01 vs EV/+PA. F, *p<0.01 vs EV/-TGF-beta-PA; #p<0.05 vs EV/+TGF-beta-PA; G, *p<0.01 vs WT.
Figure S2 A, ERK activation is responsible for FFA promotion of TGF-beta-induced inhibition of SM... more Figure S2 A, ERK activation is responsible for FFA promotion of TGF-beta-induced inhibition of SMAD4 monoubiquitination, Related to Figure 2. MDA-MB-231 cells were treated with BSA (vehicle) or PA in the presence or absence of AZD6244 or WP1130 for 4 h followed by treatment with or without 3 ng/ml of TGF-beta for another 4 h. Nuclear extracts were made and SMAD4 monoubiquitination was detected. The blot is a representative of 3 blots from 3 independent experiments. *P<0.01 vs control; #P<0.05, PA vs BSA; $P<0.01, PA+AZD vs PA, PA+WP vs PA). B, ERK activation is responsible for FFA promotion of TGF-beta-induced nuclear SAMD4 retention, Related to Figure 2. MDA-MB-231 cells were treated with 3 ng/ml of TGF-beta in the presence or absence of PA, AZD6244 or WP1130 for the times shown and processed for immunofluorescence with anti-SMAD4 antibody. The same cells were also stained with DAPI to visualize nuclei. Intensity of nuclear SMAD4 among these cells was quantified with Image-Pro Plus 6.0 software. The percentages of nuclear SMAD4 levels illustrated at the lower panel represent the mean of three independent experiments, and error bars indicate the SD.
PubMed, 2012
Purpose: To determine the prognostic value of FOXO1, GATA3 and Annexin-1 expression in breast can... more Purpose: To determine the prognostic value of FOXO1, GATA3 and Annexin-1 expression in breast cancer. Methods: Tissue microarray and individual paraffin tissue slides from 131 patients were used for the study. The association of FOXO1, GATA3 and Annexin-1 expression with clinicopathological features of breast cancer and disease outcome was examined in retrospective samples. Kaplan-Meier survival curves and Cox regression with multivariate analysis were used for assessing the relative risk (RR) and disease-free survival (DFS). The expression of FOXO1, GATA3 and Annexin-1 were determined by immunohistochemistry and the association among the three proteins was analyzed by Logistic regression analysis. Results: The nuclear expression of FOXO1 was observed in most of the normal breast tissues and 51.3% of the malignant breast tissues. GATA3 and Annexin-1 were expressed at 73% and 24.6% respectively in breast cancer tissues. The expression of FOXO1, GATA3 and Annexin-1 were all inversely correlated with lymph node-positive tumors. Both FOXO1 and Annexin-1 expression were also inversely associated with HER2-overexpressing tumors. FOXO1 expression was significantly associated with both GATA3 and Annexin-1 expression. In addition, Multivariate analyses confirm that only FOXO1 levels independently predict DFS. Conclusion: FOXO1 expression in breast cancer is regulated by the PI3K/Akt pathway. The expression of FOXO1 is also associated with GATA3 and/or Annexin-1. Restoring or targeting FOXO1 to the cell nucleus in breast cancer tissues may improve response to therapy and disease outcome. Further clinical studies are warranted to test this hypothesis.
International Journal of Oncology, Feb 5, 2015
Hormone therapy targeting estrogen receptor α (ERα) is the most effective treatment for breast ca... more Hormone therapy targeting estrogen receptor α (ERα) is the most effective treatment for breast cancer. However, this treatment eventually fails as the tumor develops resistance. Although reduced expression of ER-α is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. The present study shows that Slug has an inverse relationship with ERα in breast and prostate cancer patient samples. Also the inhibition of Slug blocks mammary stem cell activity in primary mammary epithelial cells. We hypothesize that Slug may be a key transcription factor in the regulation of ERα expression. To understand the Slug-ERα signaling pathway, we employed resistant cell line MCF-TAMR (ERα relatively negative) derived from its parental MCF-7 (ERα positive) cell line and assessed changes in cell phenotype, activity and response to therapy. Conversely, we performed knockdown of Slug in the high-Slug expressing cell line MDA-MB-231 and assessed reversal of the mesenchymal phenotype. Microarray analysis showed that Slug is overexpressed in high grade breast and prostate cancer tissues. Additionally, Slug overexpression leads to drug resistance. Furthermore, we demonstrated that Slug binds directly to ERα promoter E-boxes and represses ERα expression. This resulted in decrease in epithelial-to-mesenchymal transition in cancer cells. These findings demonstrate that Slug, by regulation of ERα expression, contributes to tumor progression and could serve as an important target for cancer therapy.
Background: Recent studies have shown that minority women, in particular African American are mor... more Background: Recent studies have shown that minority women, in particular African American are more likely to express Triple Negative (TN) tumors compared to Non-Hispanic Whites. TN tumors are more difficult to treat this may be one of the factors contributing to disparities in disease outcome and overall survival. In our study, we have studied a cohort of African American and Latina women with equal socio-economic status (SES) and access to care. The purpose of this study was to determine if these two ethnic groups with equal SES and access to care had differences in disease outcome and if this difference was related to tumor subtypes. We have also examined cellular and molecular markers associated with tumor subtypes and disease outcome. Methods: A total of 300 subjects were retrospectively selected. 156 were African American and 144 Latina. Demographic, pathologic, and clinical follow-up information were collected from existing database. The prevalence of breast cancer subtypes within racial and menopausal subsets was examined and the associations with tumor size, lymph nodal status, staging, and histologic grade were determined by logistic regression. Disease-free survival (DFS) was analyzed by Kaplan-Meier survival curves with log-rank test. Gene and protein signatures associated with PI3kinase/Akt and the CD44/CD24 expression profiles were analyzed by immunohistochemistry analysis (IHC) in different tumor subtypes. Results: The TN or basal-like breast cancer subtype (ER/PR/HER2 negative) was more prevalent among premenopausal African American women (36%) compared to postmenopausal African Americans (28.1); premenopausal Latina women (31 %); and postmenopausal Latinas (17%) (p=0.05). The HER2+/ER-subtype was more prevalent among premenopausal Latina women (17%) than postmenopausal Latinas (14%), postmenopausal African American women (9%), and premenopausal African American women (3%) (p=0.046). Prevalence of Luminal A and Luminal B breast cancer subtypes were similar with race and menopausal status. Compared to Luminal A, basal-like tumors were more poorly differentiated (OR=6.4, p<0.001) and more likely to have CD44+/CD24-phenotype, while HER2+/ER-tumors were more associated with large tumor size (OR=3.1, p=0.01) and CD24+ phenotype. The Luminal B tumors were also more likely to be associated with larger tumor size and were poorly differentiated (OR=3.0, p=0.002). Patients with basal-like and HER2+/ER-tumors had significant shorter DFS compared to patients with Luminal A tumors (log rank=11.97, p=0.0005 and log rank=8.95, p=0.002). The DFS was also reduced significantly in patients with Luminal B tumors than Luminal A tumors (log rank=7.09, p=0.007). The poor disease outcome in those patients may due to have increased plasma IGF-I, activated tissue pAkt and loss of functional FOXO1 A. Conclusions: Our study suggests a highest prevalence of basal-like tumors (triple negative) in premenopausal African American women, followed by premenopausal Latina women. Premenopausal Latina women also had a higher prevalence of HER2+/ER-tumors. DFS was poor in both ethnic groups with either basal like tumors or those with HER2+/ER-tumors. Activation of pAkt in the tumor tissue was a significant contributor to the poor disease outcome. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A96.
Figure S1 Quantification of Western blot analysis (related to Figure 1) (A-H). Data are presented... more Figure S1 Quantification of Western blot analysis (related to Figure 1) (A-H). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. *p < 0.01 versus controls; #p < 0.01 versus their corresponding DMSO controls. FFA facilitates TGF-beta-induced ERK activation and SAMD4 nuclear retention, Related to Figure 1. I, Nuclear extracts were made from MDA-MB-231 cells that were treated with 3 ng/ml of TGF-beta for the time points shown. They were analyzed by Western blotting with antibodies against Smad4 or phosphor-ERK and ERK. H3 was used as a loading control for nuclear fractions. We evaluated the cytoplasmic contamination of the nuclear fractions by blotting for α-vinculin. J, Nuclear extracts were made from MDA-MB-231 cells treated with 3 ng/ml of TGF-beta in the presence of palmitate (PA, 400 microM) for the time points shown. Phosphor-ERK and ERK and nuclear SMAD4 were analyzed by Western blotting.
International Journal of Molecular Sciences, Sep 13, 2013
Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented... more Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. B, *p<0.01 vs BSA; #p<0.01 PA+AZD vs PA. E, *p<0.01 vs EV/-PA; #p<0.01 vs EV/+PA. F, *p<0.01 vs EV/-TGF-beta-PA; #p<0.05 vs EV/+TGF-beta-PA; G, *p<0.01 vs WT.
Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. ... more Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. MDA-MB-231 cells were transfected with WT-SMAD4, HA-ubiquitin, and the indicated shRNAs and incubated with BSA or PA in the presence or absence of 3 ng/ml TGF-beta1 for 2 h. The effective knockdown of USP9x was confirmed by Western blot analysis before ubiquitination assays. Nuclear SMAD4 monoubiquitination (Mono-Ub) was detected by anti-SMAD4 immunoprecipitation and immunoblot with HA-ubiquitin. The blot is a representative of 3 blots from 3 independent experiments. *P<0.05 vs Control (shControl/-TGF-beta/-PA); #P<0.01 vs shControl/-PA; $P<0.05 shControl/+PA.
Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. H... more Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4–USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3–mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and me...
The Internet Journal of Pathology, 2009
International Journal of Clinical Investigation and Case Reports
Skene’s glands are a pair of female paraurethral glands with similar embryologic, physiologic, an... more Skene’s glands are a pair of female paraurethral glands with similar embryologic, physiologic, and pathologic features to the male prostate. Cysts and abscesses represent the most common pathological changes affecting Skene’s gland, whereas adenofibromas represent an extremely rare entity. Herein, we present a rare case report of adenofibroma in the Skene’s gland of a postmenopausal female.
One major impediment to the success of chemotherapy in cancer patients is the risk of developing ... more One major impediment to the success of chemotherapy in cancer patients is the risk of developing drug resistance. One mechanism leading to multidrug resistance in cancer therapy is through the ABC transporters. The eukaryotic ABC genes are organized as transporters containing two transmembrane and two ATP binding domains. Alternative splicing is one major mechanism in which a single gene generates functional diversity by producing different spliced isoforms. To determine the variability of ABC transporters, we performed exhaustive search of the NCBI database for ABC transporter genes, in addition to using the Alternative Splicing Annotation Project (ASAP) and the Alternative Splicing Database (ASD). We conducted immunostaining for MRP1 and MDR1 on tissue micro array of 23 breast cancer patients and RNA mediated annealing, selection, extension and ligation (RASL) assay for analysis of mRNA splicing isoforms which was performed on exon/exon target. Tumor cells from chemotherapy resistant patients show specific expression patterns of drug-resistant ATP-binding cassette transporter. Our data showed alternative splicing in 3 and 4 exon/exon borders in MDR1 and PGP genes. Two splicing events in MDR1 and one in PGP transcripts were predicted to be alternatively spliced in ASAP database. Interestingly, exon11/12 border in MDR1 is found to be alternatively spliced. In summary, protein microarray analysis demonstrated significant correlation between MRP1 expression and survival rate within 5 years. In contrast, MDRI protein expression did not correlate with 5 year survival rate. This may be due to variations in the MDR1 isoforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3531.
Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. ... more Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. MDA-MB-231 cells were transfected with WT-SMAD4, HA-ubiquitin, and the indicated shRNAs and incubated with BSA or PA in the presence or absence of 3 ng/ml TGF-beta1 for 2 h. The effective knockdown of USP9x was confirmed by Western blot analysis before ubiquitination assays. Nuclear SMAD4 monoubiquitination (Mono-Ub) was detected by anti-SMAD4 immunoprecipitation and immunoblot with HA-ubiquitin. The blot is a representative of 3 blots from 3 independent experiments. *P<0.05 vs Control (shControl/-TGF-beta/-PA); #P<0.01 vs shControl/-PA; $P<0.05 shControl/+PA.
Figure S3 FFA promotes TGF-beta-induced migration by activating ERK and USP9x, Related to Figure ... more Figure S3 FFA promotes TGF-beta-induced migration by activating ERK and USP9x, Related to Figure 3. A, BT549 cells were plated in Ibidi culture insert dishes (Madison, WI). Cells were cultured in complete medium to 80% confluence, inserts were removed to create a gap and cells were then incubated with BSA, PA, PA plus AZD6244 or WP1130 in the absence or presence of TGF-beta1. Migrating cells were photographed under a phase contrast microscope. B, The percentage of the wound closed was quantified from three independent replicates and is expressed as mean {plus minus} SD. *p<0.05 vs BSA/TGF-beta; #p<0.05 vs PA/TGF-beta.
Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented... more Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. B, *p<0.01 vs BSA; #p<0.01 PA+AZD vs PA. E, *p<0.01 vs EV/-PA; #p<0.01 vs EV/+PA. F, *p<0.01 vs EV/-TGF-beta-PA; #p<0.05 vs EV/+TGF-beta-PA; G, *p<0.01 vs WT.
Figure S2 A, ERK activation is responsible for FFA promotion of TGF-beta-induced inhibition of SM... more Figure S2 A, ERK activation is responsible for FFA promotion of TGF-beta-induced inhibition of SMAD4 monoubiquitination, Related to Figure 2. MDA-MB-231 cells were treated with BSA (vehicle) or PA in the presence or absence of AZD6244 or WP1130 for 4 h followed by treatment with or without 3 ng/ml of TGF-beta for another 4 h. Nuclear extracts were made and SMAD4 monoubiquitination was detected. The blot is a representative of 3 blots from 3 independent experiments. *P<0.01 vs control; #P<0.05, PA vs BSA; $P<0.01, PA+AZD vs PA, PA+WP vs PA). B, ERK activation is responsible for FFA promotion of TGF-beta-induced nuclear SAMD4 retention, Related to Figure 2. MDA-MB-231 cells were treated with 3 ng/ml of TGF-beta in the presence or absence of PA, AZD6244 or WP1130 for the times shown and processed for immunofluorescence with anti-SMAD4 antibody. The same cells were also stained with DAPI to visualize nuclei. Intensity of nuclear SMAD4 among these cells was quantified with Image-Pro Plus 6.0 software. The percentages of nuclear SMAD4 levels illustrated at the lower panel represent the mean of three independent experiments, and error bars indicate the SD.
PubMed, 2012
Purpose: To determine the prognostic value of FOXO1, GATA3 and Annexin-1 expression in breast can... more Purpose: To determine the prognostic value of FOXO1, GATA3 and Annexin-1 expression in breast cancer. Methods: Tissue microarray and individual paraffin tissue slides from 131 patients were used for the study. The association of FOXO1, GATA3 and Annexin-1 expression with clinicopathological features of breast cancer and disease outcome was examined in retrospective samples. Kaplan-Meier survival curves and Cox regression with multivariate analysis were used for assessing the relative risk (RR) and disease-free survival (DFS). The expression of FOXO1, GATA3 and Annexin-1 were determined by immunohistochemistry and the association among the three proteins was analyzed by Logistic regression analysis. Results: The nuclear expression of FOXO1 was observed in most of the normal breast tissues and 51.3% of the malignant breast tissues. GATA3 and Annexin-1 were expressed at 73% and 24.6% respectively in breast cancer tissues. The expression of FOXO1, GATA3 and Annexin-1 were all inversely correlated with lymph node-positive tumors. Both FOXO1 and Annexin-1 expression were also inversely associated with HER2-overexpressing tumors. FOXO1 expression was significantly associated with both GATA3 and Annexin-1 expression. In addition, Multivariate analyses confirm that only FOXO1 levels independently predict DFS. Conclusion: FOXO1 expression in breast cancer is regulated by the PI3K/Akt pathway. The expression of FOXO1 is also associated with GATA3 and/or Annexin-1. Restoring or targeting FOXO1 to the cell nucleus in breast cancer tissues may improve response to therapy and disease outcome. Further clinical studies are warranted to test this hypothesis.
International Journal of Oncology, Feb 5, 2015
Hormone therapy targeting estrogen receptor α (ERα) is the most effective treatment for breast ca... more Hormone therapy targeting estrogen receptor α (ERα) is the most effective treatment for breast cancer. However, this treatment eventually fails as the tumor develops resistance. Although reduced expression of ER-α is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. The present study shows that Slug has an inverse relationship with ERα in breast and prostate cancer patient samples. Also the inhibition of Slug blocks mammary stem cell activity in primary mammary epithelial cells. We hypothesize that Slug may be a key transcription factor in the regulation of ERα expression. To understand the Slug-ERα signaling pathway, we employed resistant cell line MCF-TAMR (ERα relatively negative) derived from its parental MCF-7 (ERα positive) cell line and assessed changes in cell phenotype, activity and response to therapy. Conversely, we performed knockdown of Slug in the high-Slug expressing cell line MDA-MB-231 and assessed reversal of the mesenchymal phenotype. Microarray analysis showed that Slug is overexpressed in high grade breast and prostate cancer tissues. Additionally, Slug overexpression leads to drug resistance. Furthermore, we demonstrated that Slug binds directly to ERα promoter E-boxes and represses ERα expression. This resulted in decrease in epithelial-to-mesenchymal transition in cancer cells. These findings demonstrate that Slug, by regulation of ERα expression, contributes to tumor progression and could serve as an important target for cancer therapy.
Background: Recent studies have shown that minority women, in particular African American are mor... more Background: Recent studies have shown that minority women, in particular African American are more likely to express Triple Negative (TN) tumors compared to Non-Hispanic Whites. TN tumors are more difficult to treat this may be one of the factors contributing to disparities in disease outcome and overall survival. In our study, we have studied a cohort of African American and Latina women with equal socio-economic status (SES) and access to care. The purpose of this study was to determine if these two ethnic groups with equal SES and access to care had differences in disease outcome and if this difference was related to tumor subtypes. We have also examined cellular and molecular markers associated with tumor subtypes and disease outcome. Methods: A total of 300 subjects were retrospectively selected. 156 were African American and 144 Latina. Demographic, pathologic, and clinical follow-up information were collected from existing database. The prevalence of breast cancer subtypes within racial and menopausal subsets was examined and the associations with tumor size, lymph nodal status, staging, and histologic grade were determined by logistic regression. Disease-free survival (DFS) was analyzed by Kaplan-Meier survival curves with log-rank test. Gene and protein signatures associated with PI3kinase/Akt and the CD44/CD24 expression profiles were analyzed by immunohistochemistry analysis (IHC) in different tumor subtypes. Results: The TN or basal-like breast cancer subtype (ER/PR/HER2 negative) was more prevalent among premenopausal African American women (36%) compared to postmenopausal African Americans (28.1); premenopausal Latina women (31 %); and postmenopausal Latinas (17%) (p=0.05). The HER2+/ER-subtype was more prevalent among premenopausal Latina women (17%) than postmenopausal Latinas (14%), postmenopausal African American women (9%), and premenopausal African American women (3%) (p=0.046). Prevalence of Luminal A and Luminal B breast cancer subtypes were similar with race and menopausal status. Compared to Luminal A, basal-like tumors were more poorly differentiated (OR=6.4, p<0.001) and more likely to have CD44+/CD24-phenotype, while HER2+/ER-tumors were more associated with large tumor size (OR=3.1, p=0.01) and CD24+ phenotype. The Luminal B tumors were also more likely to be associated with larger tumor size and were poorly differentiated (OR=3.0, p=0.002). Patients with basal-like and HER2+/ER-tumors had significant shorter DFS compared to patients with Luminal A tumors (log rank=11.97, p=0.0005 and log rank=8.95, p=0.002). The DFS was also reduced significantly in patients with Luminal B tumors than Luminal A tumors (log rank=7.09, p=0.007). The poor disease outcome in those patients may due to have increased plasma IGF-I, activated tissue pAkt and loss of functional FOXO1 A. Conclusions: Our study suggests a highest prevalence of basal-like tumors (triple negative) in premenopausal African American women, followed by premenopausal Latina women. Premenopausal Latina women also had a higher prevalence of HER2+/ER-tumors. DFS was poor in both ethnic groups with either basal like tumors or those with HER2+/ER-tumors. Activation of pAkt in the tumor tissue was a significant contributor to the poor disease outcome. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A96.
Figure S1 Quantification of Western blot analysis (related to Figure 1) (A-H). Data are presented... more Figure S1 Quantification of Western blot analysis (related to Figure 1) (A-H). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. *p < 0.01 versus controls; #p < 0.01 versus their corresponding DMSO controls. FFA facilitates TGF-beta-induced ERK activation and SAMD4 nuclear retention, Related to Figure 1. I, Nuclear extracts were made from MDA-MB-231 cells that were treated with 3 ng/ml of TGF-beta for the time points shown. They were analyzed by Western blotting with antibodies against Smad4 or phosphor-ERK and ERK. H3 was used as a loading control for nuclear fractions. We evaluated the cytoplasmic contamination of the nuclear fractions by blotting for α-vinculin. J, Nuclear extracts were made from MDA-MB-231 cells treated with 3 ng/ml of TGF-beta in the presence of palmitate (PA, 400 microM) for the time points shown. Phosphor-ERK and ERK and nuclear SMAD4 were analyzed by Western blotting.
International Journal of Molecular Sciences, Sep 13, 2013
Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented... more Figure S4 Quantification of Western blot analysis (related to Figure 4) (B-G). Data are presented as mean fold increases ({plus minus}SD) in treated groups over basal values from three independent experiments. B, *p<0.01 vs BSA; #p<0.01 PA+AZD vs PA. E, *p<0.01 vs EV/-PA; #p<0.01 vs EV/+PA. F, *p<0.01 vs EV/-TGF-beta-PA; #p<0.05 vs EV/+TGF-beta-PA; G, *p<0.01 vs WT.
Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. ... more Figure S5 USP9x is responsible for TGF-beta-induced SMAD4 deubiquitination, Related to Figure 5. MDA-MB-231 cells were transfected with WT-SMAD4, HA-ubiquitin, and the indicated shRNAs and incubated with BSA or PA in the presence or absence of 3 ng/ml TGF-beta1 for 2 h. The effective knockdown of USP9x was confirmed by Western blot analysis before ubiquitination assays. Nuclear SMAD4 monoubiquitination (Mono-Ub) was detected by anti-SMAD4 immunoprecipitation and immunoblot with HA-ubiquitin. The blot is a representative of 3 blots from 3 independent experiments. *P<0.05 vs Control (shControl/-TGF-beta/-PA); #P<0.01 vs shControl/-PA; $P<0.05 shControl/+PA.
Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. H... more Obesity increases the risk of distant metastatic recurrence and reduces breast cancer survival. However, the mechanisms behind this pathology and identification of relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are elevated in obese individuals. Here we report that TGFβ transiently activates ERK and subsequently phosphorylates SMAD4 at Thr277, which facilitates a SMAD4–USP9x interaction, SMAD4 nuclear retention, and stimulates TGFβ/SMAD3–mediated transcription of Twist and Snail. USP9x inhibited the E3 ubiquitin-protein ligase TIF1γ from binding and monoubiquitinating SMAD4, hence maintaining the SMAD4 nuclear retention. FFA further facilitated TGFβ-induced ERK activation, SMAD4 phosphorylation, and nuclear retention, promoting TGFβ-dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. In addition, clinical data indicated that phospho-ERK and -SMAD4 levels correlate with activated TGFβ signaling and me...