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Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion.

Elsarraj HS, Hong Y, Valdez KE, Michaels W, Hook M, Smith WP, Chien J, Herschkowitz JI, Troester MA, Beck M, Inciardi M, Gatewood J, May L, Cusick T, McGinness M, Ricci L, Fan F, Tawfik O, Marks JR, Knapp JR, Yeh HW, Thomas P, Carrasco DR, Fields TA, Godwin AK, Behbod F - Breast Cancer Res. (2015)

Bottom Line: BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription.This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %).A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Kansas Medical Center, 3901 Rainbow Blvd, Mail Stop 3003, Kansas City, KS, 66160, USA. helsarraj@kumc.edu.

ABSTRACT

Introduction: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.

Methods: Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.

Results: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.

Conclusion: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

No MeSH data available.


Related in: MedlinePlus

Enhanced BCL9 nuclear expression in DCIS cell line MIND xenografts that progressed to invasive lesions. a, b RT-qPCR of BCL9 in epithelial cell adhesion molecule (EpCAM)-positive epithelial cells sorted from SUM225 (a) and DCIS.COM (b) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection. Bar graphs represent fold change normalized to 2 weeks. Data are mean values ± standard error of the mean (n = 3, *p <0.05). c, d Immunofluorescent (IF) staining of BCL9, keratin (K)5/K19, and 4′,6-diamidino-2-phenylindole (DAPI) in SUM225 (c) and DCIS.COM (d) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection; merged channels are shown in the upper panels and BCL9-only channels are shown in the lower panel. BCL9 was conjugated to Alexa-Fluor 594 (red) and keratin 5/keratin 19 were conjugated to Alexa-Fluor 488 (green). Nuclei were counterstained with DAPI. Scale bars are 50 μm; × 40 objective was used
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Fig3: Enhanced BCL9 nuclear expression in DCIS cell line MIND xenografts that progressed to invasive lesions. a, b RT-qPCR of BCL9 in epithelial cell adhesion molecule (EpCAM)-positive epithelial cells sorted from SUM225 (a) and DCIS.COM (b) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection. Bar graphs represent fold change normalized to 2 weeks. Data are mean values ± standard error of the mean (n = 3, *p <0.05). c, d Immunofluorescent (IF) staining of BCL9, keratin (K)5/K19, and 4′,6-diamidino-2-phenylindole (DAPI) in SUM225 (c) and DCIS.COM (d) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection; merged channels are shown in the upper panels and BCL9-only channels are shown in the lower panel. BCL9 was conjugated to Alexa-Fluor 594 (red) and keratin 5/keratin 19 were conjugated to Alexa-Fluor 488 (green). Nuclei were counterstained with DAPI. Scale bars are 50 μm; × 40 objective was used

Mentions: To confirm our microarray analysis, RT-qPCR was performed on EpCAM-positive cells sorted from an independent set of DCIS cell line MIND xenografts as they progressed from 2 to 10 weeks. BCL9 gene expression was significantly increased at 10 compared to 2 weeks in both SUM225 and DCIS.COM MIND xenografts (62 ± 14 and 35 ± 12 fold increase, respectively; mean ± SEM p <0.05) (Fig. 3a, b). Furthermore, IF staining of the MIND xenografts demonstrated increased nuclear BCL9 expression as DCIS lesions progressed to invasion (Fig. 3c, d). There have been a few reports on the role of BCL9L (BCL9-2 or B9L), BCL9 homolog, in breast cancer. One study showed nuclear BCL9L expression to be significantly associated with high nuclear grade and the expression of HER2 in breast cancers [36]. Another study reported that BCL9L induced ER positive breast cancers in vivo by regulating the expression of ER through a β-catenin independent mechanism and predicted therapeutic response to tamoxifen [37]. The human BCL9 and its homolog BCL9L reside on chromosome 1q21 and 11q23.3 respectively. Both BCL9 and BCL9L have been shown to function as co-activators of β-catenin-LEF/TCF mediated transcription [38, 39]. We compared the expression patterns of BCL9 and BCL9L in DCIS cell line MIND xenografts and on tissue sections obtained from 23 patients with DCIS and associated IDC and 14 patients with pure DCIS. Additional file 5: Figure S2A shows that BCL9L expression was mainly cytoplasmic, while BCL9 expression was primarily nuclear. Furthermore, RT-qPCR showed no significant increase in BCL9L expression in DCIS MIND xenografts during invasive transition from 2 to 10 weeks (Additional file 5: Figure S2B-C). Western blot on cell lysates obtained from DCIS cell lines also showed no change in BCL9L expression with BCL9 KD. Therefore, the results in both the MIND and tandem lesions support the hypothesis that increased BCL9 expression is associated with DCIS transition to invasion, while our data do not show a change in BCL9L expression associated with DCIS progression.Fig. 3


Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion.

Elsarraj HS, Hong Y, Valdez KE, Michaels W, Hook M, Smith WP, Chien J, Herschkowitz JI, Troester MA, Beck M, Inciardi M, Gatewood J, May L, Cusick T, McGinness M, Ricci L, Fan F, Tawfik O, Marks JR, Knapp JR, Yeh HW, Thomas P, Carrasco DR, Fields TA, Godwin AK, Behbod F - Breast Cancer Res. (2015)

Enhanced BCL9 nuclear expression in DCIS cell line MIND xenografts that progressed to invasive lesions. a, b RT-qPCR of BCL9 in epithelial cell adhesion molecule (EpCAM)-positive epithelial cells sorted from SUM225 (a) and DCIS.COM (b) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection. Bar graphs represent fold change normalized to 2 weeks. Data are mean values ± standard error of the mean (n = 3, *p <0.05). c, d Immunofluorescent (IF) staining of BCL9, keratin (K)5/K19, and 4′,6-diamidino-2-phenylindole (DAPI) in SUM225 (c) and DCIS.COM (d) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection; merged channels are shown in the upper panels and BCL9-only channels are shown in the lower panel. BCL9 was conjugated to Alexa-Fluor 594 (red) and keratin 5/keratin 19 were conjugated to Alexa-Fluor 488 (green). Nuclei were counterstained with DAPI. Scale bars are 50 μm; × 40 objective was used
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Enhanced BCL9 nuclear expression in DCIS cell line MIND xenografts that progressed to invasive lesions. a, b RT-qPCR of BCL9 in epithelial cell adhesion molecule (EpCAM)-positive epithelial cells sorted from SUM225 (a) and DCIS.COM (b) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection. Bar graphs represent fold change normalized to 2 weeks. Data are mean values ± standard error of the mean (n = 3, *p <0.05). c, d Immunofluorescent (IF) staining of BCL9, keratin (K)5/K19, and 4′,6-diamidino-2-phenylindole (DAPI) in SUM225 (c) and DCIS.COM (d) MIND xenografts at 2, 6, and 10 weeks post-intraductal injection; merged channels are shown in the upper panels and BCL9-only channels are shown in the lower panel. BCL9 was conjugated to Alexa-Fluor 594 (red) and keratin 5/keratin 19 were conjugated to Alexa-Fluor 488 (green). Nuclei were counterstained with DAPI. Scale bars are 50 μm; × 40 objective was used
Mentions: To confirm our microarray analysis, RT-qPCR was performed on EpCAM-positive cells sorted from an independent set of DCIS cell line MIND xenografts as they progressed from 2 to 10 weeks. BCL9 gene expression was significantly increased at 10 compared to 2 weeks in both SUM225 and DCIS.COM MIND xenografts (62 ± 14 and 35 ± 12 fold increase, respectively; mean ± SEM p <0.05) (Fig. 3a, b). Furthermore, IF staining of the MIND xenografts demonstrated increased nuclear BCL9 expression as DCIS lesions progressed to invasion (Fig. 3c, d). There have been a few reports on the role of BCL9L (BCL9-2 or B9L), BCL9 homolog, in breast cancer. One study showed nuclear BCL9L expression to be significantly associated with high nuclear grade and the expression of HER2 in breast cancers [36]. Another study reported that BCL9L induced ER positive breast cancers in vivo by regulating the expression of ER through a β-catenin independent mechanism and predicted therapeutic response to tamoxifen [37]. The human BCL9 and its homolog BCL9L reside on chromosome 1q21 and 11q23.3 respectively. Both BCL9 and BCL9L have been shown to function as co-activators of β-catenin-LEF/TCF mediated transcription [38, 39]. We compared the expression patterns of BCL9 and BCL9L in DCIS cell line MIND xenografts and on tissue sections obtained from 23 patients with DCIS and associated IDC and 14 patients with pure DCIS. Additional file 5: Figure S2A shows that BCL9L expression was mainly cytoplasmic, while BCL9 expression was primarily nuclear. Furthermore, RT-qPCR showed no significant increase in BCL9L expression in DCIS MIND xenografts during invasive transition from 2 to 10 weeks (Additional file 5: Figure S2B-C). Western blot on cell lysates obtained from DCIS cell lines also showed no change in BCL9L expression with BCL9 KD. Therefore, the results in both the MIND and tandem lesions support the hypothesis that increased BCL9 expression is associated with DCIS transition to invasion, while our data do not show a change in BCL9L expression associated with DCIS progression.Fig. 3

Bottom Line: BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription.This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %).A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Kansas Medical Center, 3901 Rainbow Blvd, Mail Stop 3003, Kansas City, KS, 66160, USA. helsarraj@kumc.edu.

ABSTRACT

Introduction: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.

Methods: Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.

Results: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.

Conclusion: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

No MeSH data available.


Related in: MedlinePlus