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. 2013 May 23;32(21):2631-9.
doi: 10.1038/onc.2012.286. Epub 2012 Jul 9.

Regulation of DCIS to invasive breast cancer progression by Singleminded-2s (SIM2s)

K C Scribner  1 , F Behbod, W W Porter
Affiliations

Regulation of DCIS to invasive breast cancer progression by Singleminded-2s (SIM2s)

K C Scribner et al. Oncogene. .

Abstract

Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of mammary epithelial cell differentiation. SIM2s is highly expressed in mammary epithelial cells and downregulated in human breast cancer. Loss of Sim2s causes aberrant mouse mammary ductal development, with features suggestive of malignant transformation, whereas overexpression of SIM2s promotes precocious alveolar differentiation in nulliparous mouse mammary glands, suggesting that SIM2s is required for establishing and enhancing mammary gland differentiation. To test the hypothesis that SIM2s regulates tumor cell differentiation, we analyzed SIM2s expression in human primary breast ductal carcinoma in situ (DCIS) samples and found that SIM2s is lost with progression from DCIS to invasive ductal cancer (IDC). Using a MCF10DCIS.COM progression model, we have shown that SIM2s expression is decreased in MCF10DCIS.COM cells compared with MCF10A cells, and reestablishment of SIM2s in MCF10DCIS.COM cells significantly inhibits growth and invasion both in vitro and in vivo. Analysis of SIM2s-MCF10DCIS.com tumors showed that SIM2s promoted a more differentiated tumor phenotype including the expression of a broad range of luminal markers (CSN2 (β-casein), CDH1 (E-cadherin), and KER18 (keratin-18)) and suppressed genes associated with stem cell maintenance and a basal phenotype (SMO (smoothened), p63, SLUG (snail-2), KER14 (keratin-14) and VIM (vimentin)). Furthermore, loss of SIM2s expression in MCF10DCIS.COM xenografts resulted in a more invasive phenotype and increased lung metastasis likely due to an increase in Hedgehog signaling and matrix metalloproteinase expression. Together, these exciting new data support a role for SIM2s in promoting human breast tumor differentiation and maintaining epithelial integrity.

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Conflict of interest statement

Conflict of Interest

The authors have nothing to disclose.

Figures

Figure 1
Figure 1. SIM2s expression is progressively lost in human ductal carcinoma in situ (DCIS) transition to invasive ductal cancer (IDC)
A – Human Normal-like tissue, B – Human DCIS, C – Human IDC. Top Row – H&E staining of Normal, DCIS, and IDC samples. Bottom Rows - SIM2s immunohistochemistry of Normal, DCIS and IDC samples. Normal-like structures show clean, punctate nuclear staining. DCIS samples show nuclear and cytoplasmic SIM2s staining in over 75% of samples, and this expression is lost in over 80% of IDC samples. Images were taken at 10x and 40x objective (6.3x and 25.2x), scale bars represent 100μm. An n=14 was used for each tumor classification (DCIS and IDC).
Figure 2
Figure 2. Analysis of MCF10DCIS cell transductions in vitro show changes in proliferation, invasion, and differentiation markers
A – Q-PCR analysis of SIM2s mRNA levels in MCF10DCIS.COM and parent MCF10A cells, as well as commonly used breast cancer cell lines MCF7 and MDA.MB.231. B, C - Immunofluorescent staining of SIM2s to confirm nuclear SIM2s overexpression. D – Q-PCR analysis of SIM2s in the MCF10DCIS.com cell line confirming an approximate 80% loss of expression in adhered SIM2si cells. E – Proliferation assays confirm that SIM2s expression inhibits breast cancer cell proliferation, while loss of SIM2s increases growth. The values shown are the mean ± SE of triplicate samples. F - Boyden chamber invasion assay shows significantly more SIM2si cells were able to invade and migrate compared to controls. Values are the average number of cells per five fields per membrane of three separate plates. G & H - Q-PCR analysis of differentiation markers CDH1 and p21. * = p-value < .05.
Figure 3
Figure 3. Differential SIM2s expression regulates growth in vivo.
A & B – Analysis of xenograft tumor growth and mass shows that over-expression and loss of SIM2s expression inhibits xenograft growth. C – Q-PCR analysis of SIM2s mRNA expression in SIM2si tumors shows an approximate 50% loss of expression in vivo. D – H&E histological analysis shows that SIM2s expressing tumors exhibit smaller, more lobulo-like structures throughout the tumor, with less necrosis and inflammation. Images were taken using a 5x objective (5x) 10x objective (6.3x) and a 40x objective (25.2x). Scale bars represent 100μm. Arrows indicate areas of invasion. D – Histological analysis of SIM2s expression in xenografts confirms SIM2s overexpression and loss of expression. Images were taken using a 40x objective (25.2x). Scale bars represent 100μm. * = p-value < .05.
Figure 4
Figure 4. SIM2s decreases markers associated with basal breast cancer in MCF10DCIS.COM xenografts
A, B, C, & D – Immunohistochemical staining for basal markers including KER14, SMA, VIM, and p63. Images were taken using a 40x objective (25.2x). Scale bars represent 100μm. E – KER5/VIM immunofluorescence shows increased overlap of KER5 and VIM in SIM2si xenografts, which is associated with enhanced invasive potential and aggressiveness. F, G, H, & I – Q-PCR analysis of basal markers SLUG, SMA, and p63, as well as cell cycle regulator p21. * = p-value < .05.
Figure 5
Figure 5. SIM2s xenografts have increased levels of luminal markers and express β-Casein
A, B, & C –Re-establishment of SIM2s promotes apical localization of CDH1 and increased KER18 expression, while SIM2s loss results in a decrease in expression. SIM2s expression also promotes apical expression of luminal marker MUC-1, whereas loss of SIM2s causes a loss of localization. D, E, F, & G – Q-PCR analysis of luminal markers CDH1, KER18, CSN2, and GATA3. H – Immunohistochemical analysis for CSN2 shows elevated levels in SIM2s tumors (see arrows). Images were taken using a 40x objective (25.2x) and 63x objective (63x). Scale bars of images represent 100μm. * = p-value < .05.
Figure 6
Figure 6. SIM2s inhibits metastasis and alters angiogenesis
A – Immunohistochemical analysis of lung tissue for vimentin (VIM) positive micrometastases showed decreased staining in SIM2s tumors, whereas loss of SIM2s enhanced lung metastasis. Images were taken with a 40x objective (25.2x). B – Q-PCR analysis for human β-2-Globulin as an indicator of lung metastasis confirmed the effect of SIM2s on metastasis with a decreased in β-2-Globulin expression in SIM2s tumors and increased expression with loss of SIM2s. Data is shown as the number of β-2G positive samples out of the total number of samples analyzed. C – Increased magnification (63x) of VIM staining to indicate the presence of vimentin positive cells in Scrambled controls and SIM2si tumors. D – Immunostaining for CD31 expression showed that SIM2s tumors have smaller blood vessels that remained on the outer perimeter of the tumors, whereas SIM2si tumors had an increased trend in angiogenesis. E – Quantification of CD31 staining by measuring blood vessel length confirms trends seen with CD31 immunohistochemistry. Images were taken using a 10x objective (6.3x) and a 40x objective (25.2x). Scale bars in images represent 100μm. *= p-value < .05
Figure 7
Figure 7. Loss of SIM2s increases tumor invasiveness through MMP expression and Hedgehog signaling
A, B, C & D – Q-PCR analysis of various MMPs showing a decrease in expression with SIM2s tumors and an increase in SIM2si tumors, a likely mechanism for increased tumor invasiveness and metastasis. E & F – Q-PCR analysis of Indian Hedgehog (IHH) and Smoothened (SMO) show elevated levels of expression in SIM2si tumors. * - p-value < .05.
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