Analysis of the Transcriptome: Regulation of Cancer Stemness in Breast Ductal Carcinoma In Situ by Vitamin D Compounds.

Ductal carcinoma in situ (DCIS), which accounts for one out of every five new breast cancer diagnoses, will progress to potentially lethal invasive ductal carcinoma (IDC) in about 50% of cases. Vitamin D compounds have been shown to inhibit progression to IDC in the MCF10DCIS model. This inhibition appears to involve a reduction in the cancer stem cell-like population in MCF10DCIS tumors. To identify genes that are involved in the vitamin D effects, a global transcriptomic analysis was undertaken of MCF10DCIS cells grown in mammosphere cultures, in which cancer stem-like cells grow preferentially and produce colonies by self-renewal and maturation, in the presence and absence of 1α25(OH)2D3 and a vitamin D analog, BXL0124. Using next-generation RNA-sequencing, we found that vitamin D compounds downregulated genes involved in maintenance of breast cancer stem-like cells (e.g., GDF15), epithelial-mesenchymal transition, invasion, and metastasis (e.g., LCN2 and S100A4), and chemoresistance (e.g., NGFR, PPP1R1B, and AGR2), while upregulating genes associated with a basal-like phenotype (e.g., KRT6A and KRT5) and negative regulators of breast tumorigenesis (e.g., EMP1). Gene methylation status was analyzed to determine whether the changes in expression induced by vitamin D compounds occurred via this mechanism. Ingenuity pathway analysis was performed to identify upstream regulators and downstream signaling pathway genes differentially regulated by vitamin D, including TP63 and vitamin D receptor -mediated canonical pathways in particular. This study provides a global profiling of changes in the gene signature of DCIS regulated by vitamin D compounds and possible targets for chemoprevention of DCIS progression to IDC in patients.

Shan NL ，Minden A ，Furmanski P ，Bak MJ ，Cai L ，Wernyj R ，Sargsyan D ，Cheng D ，Wu R ，Kuo HD ，Li SN ，Fang M ，Maehr H ，Kong AN ，Suh N ... -  《-》

Inhibition of the transition of ductal carcinoma in situ to invasive ductal carcinoma by a Gemini vitamin D analog.

Ductal carcinoma in situ (DCIS) is a nonmalignant lesion of the breast with the potential to progress to invasive ductal carcinoma (IDC). The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS have been identified as major events in the development of breast cancer. The MCF10DCIS.com cell line is a well-established model, which recapitulates the progression of breast cancer from DCIS to IDC. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) is a potent inhibitor of the growth of MCF10DCIS.com xenografted tumors without hypercalcemic toxicity. In this study, we utilized the MCF10DCIS.com in vivo model to assess the effects of BXL0124 on breast cancer progression from weeks 1 to 4. Upon DCIS progression to IDC from weeks 3 to 4, tumors lost the myoepithelial cell layer and basement membrane as shown by immunofluorescence staining with smooth muscle actin and laminin 5, respectively. Administration of BXL0124 maintained the critical myoepithelial cell layer as well as basement membrane, and animals treated with BXL0124 showed a 43% reduction in tumor volume by week 4. BXL0124 treatment decreased cell proliferation and maintained vitamin D receptor levels in tumors. In addition, the BXL0124 treatment reduced the mRNA levels of matrix metalloproteinases starting at week 3, contributing to the inhibition of invasive transition. Our results suggest that the maintenance of DCIS plays a significant role in the cancer preventive action of the Gemini vitamin D BXL0124 during the progression of breast lesions.

Wahler J ，So JY ，Kim YC ，Liu F ，Maehr H ，Uskokovic M ，Suh N ... -  《-》

Vitamin D compounds reduce mammosphere formation and decrease expression of putative stem cell markers in breast cancer.

Breast cancer stem cells (BCSCs) are a subset of tumor cells that are believed to be the cells responsible for the establishment and maintenance of tumors. Moreover, BCSCs are suggested to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). We have previously reported that a vitamin D compound, BXL0124, inhibits the progression of DCIS to IDC. In the present study we sought to determine whether this effect was mediated through an influence on BCSCs. In MCF10DCIS cells treated with vitamin D compounds (1α25(OH)2D3 or BXL0124), the breast cancer stem cell-like population, identified by the CD44(+)/CD24(-/low) and CD49f(+)/CD24(-/low) subpopulations, was reduced. To determine the effects of vitamin D compounds on cancer stem cell activity, the MCF10DCIS mammosphere cell culture system, which enriches for mammary progenitor cells and putative BCSCs, was utilized. Untreated MCF10DCIS mammospheres showed a disorganized and irregular shape. When MCF10DCIS cells were treated with 1α25(OH)2D3 or BXL0124, the mammospheres that formed exhibited a more organized, symmetrical and circular shape, similar to the appearance of spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. The mammosphere forming efficiency (MFE) was significantly decreased upon treatment with 1α25(OH)2D3 or BXL0124, indicating that these compounds have an inhibitory effect on mammosphere development. Treatment with 1α25(OH)2D3 or BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, CD49f, c-Notch1, and pNFκB. Furthermore, 1α25(OH)2D3 and BXL0124 reduced the expression of pluripotency markers, OCT4 and KLF-4 in mammospheres. This study suggests that vitamin D compounds repress the breast cancer stem cell-like population, potentially contributing to their inhibition of breast cancer. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Wahler J ，So JY ，Cheng LC ，Maehr H ，Uskokovic M ，Suh N ... -  《-》

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 ... -  《-》

Silencing of HSulf-2 expression in MCF10DCIS.com cells attenuate ductal carcinoma in situ progression to invasive ductal carcinoma in vivo.

Khurana A ，McKean H ，Kim H ，Kim SH ，mcguire J ，Roberts LR ，Goetz MP ，Shridhar V ... -  《-》