The adaptor SASH1 acts through NOTCH1 and its inhibitor DLK1 in a 3D model of lumenogenesis involving CEACAM1

Abstract

CEACAM1 transfection into breast cancer cells restores lumen formation in a 3D culture model. Among the top up-regulated genes that were associated with restoration of lumen formation, the adaptor protein SASH1 was identified. Furthermore, SASH1 was shown to be critical for lumen formation by RNAi inhibition. Upon analyzing the gene array from CEACAM1/MCF7 cells treated with SASH1 RNAi, DLK1, an inhibitor of NOTCH1 signaling, was found to be down-regulated to the same extent as SASH1. Subsequent treatment of CEACAM1/MCF7 cells with RNAi to DLK1 also inhibited lumen formation, supporting its association with SASH1. In agreement with the role of DLK1 as a NOTCH1 inhibitor, NOTCH1, as well as its regulated genes HES1 and HEY1, were down-regulated in CEACAM1/MCF7 cells by the action of DLK1 RNAi, and up-regulated by SASH1 RNAi. When CEACAM1/MCF7 cells were treated with a γ-secretase inhibitor known to inhibit NOTCH signaling, lumen formation was inhibited. We conclude that restoration of lumen formation by CEACAM1 regulates the NOTCH1 signaling pathway via the adaptor protein SASH1 and the NOTCH1 inhibitor DLK1. These data suggest that the putative involvement of NOTCH1 as a tumor-promoting gene in breast cancer may depend on its lack of regulation in cancer, whereas its involvement in normal lumen formation requires activation of its expression, and subsequently, inhibition of its signaling.

Keywords: Breast cancer; CEACAM1; DLK1; Lumeogenesis; NOTCH1; SASH1.

Figure 1:. Sash1 is up-regulated by CEACAM1 expression and is critical for lumenogenesis.

(A) MCF7 parental cells (WT) and CEACAM1/MCF7 cells (CEACAM1) were grown in 3D culture for six days, proteins separated by SDS gel electrophoresis and analyzed by western blotting for SASH1 and β-actin. (B) mRNA expression of SASH1 in CEACAM1/MCF7 cells untreated, treated with lipofectamine (lipo), treated with a negative, scrambled oligo control (ctrl) or treated with SASH1 specific RNAi. mRNA levels were quantified by qRT-PCR in triplicate. (C) CEACAM1/MCF7 cells treated with controls or with SASH1 RNAi were grown on Matrigel for 6 days and lumen were examined at 50x by phase-contrast microscopy (n= 200, P= 0.001, for RNAi treated vs transfectamine control). Magnification 40x, inset 80x. Acini counted had the following dimensions: D= 40–60-μm, with lumen size D= 8–10 μm.

Figure 2:. Inhibition of SASH1 expression by RNAi inhibits DLK1 expression.

(A) mRNA expression of DLK1 in CEACAM1/MCF7 cells untreated (None), treated with a negative, scrambled oligo control (Ctrl) or treated with SASH1 specific RNAi at 10 nM or 20 nM concentrations. mRNA levels were quantified by qRT-PCR in triplicate. (B) CEACAM1/MCF7 cells were untreated (None), treated with lipofectamine (Lipo), treated with a negative, scrambled oligo control (Ctrl), or treated with SASH1 RNAi for three days and proteins analyzed by SDS gel electrophoresis followed by western blotting for DLK1 and β-actin. Values normalized for equal protein loading are shown. (C) mRNA expression of DLK1 in CEACAM1/MCF7 cells treated with lipofectamine (Lipo), a negative, scrambled oligo control (Ctrl) or DLK1 RNAi for three days was quantified by qRT-PCR in triplicate. (D) mRNA expression of SASH1 in CEACAM1/MCF7 cells untreated (None), treated with lipofectamine (Lipo), treated with a negative, scrambled oligo control (Ctrl) or treated with DLK1 specific RNAi. mRNA levels were quantified by qRT-PCR in triplicate (**, p<0.001).

Figure 3:. Inhibition of SASH1 by RNAi downregulates both DLK1 and DLK2.

(A) mRNA levels of DLK2 compared to DLK1 by qRT-PCR in triplicate in CEACAM1/MCF7 cells. (B) mRNA levels of DLK2 from untreated or DLK1 RNAi treated CEACAM1/MCF7 cells. (C) mRNA levels of DLK2 by qRT-PCR in triplicate from untreated or SASH1 RNAi treated CEACAM1/MCF7 cells at two different concentrations (10 nM and 20 nM) after growth in 3D culture for six days. (D) DLK2 mRNA expression by qRT-PCR in triplicate from CEACAM1/MCF7 cells over expressing SASH1-GFP compared to the non-transfected control.

Figure 4:. Inhibition of both DLK1 and DLK2 are required for inhibition of lumen formation.

(A) mRNA expression of DLK1 in CEACAM1/MCF7 cells untreated (No Tx), treated with control RNAi (Ctrl), or DLK1 RNAi 1 through 4 (1 and 2 from Origene; 3 and 4 from Ambion). (B) mRNA expression of DLK2 in CEACAM1/MCF7 cells untreated (No Tx), treated with control RNAi (Ctrl), or treated with DLK1 RNAs 1 through 4 as above. (C) Quantitation of lumen formation of CEACAM1/MCF7 cells treated with RNAi 1–4 and grown in 3D vs no treatment (None) or lipofectamine (Lipo). (D) Representative micrographs (low resolution and inset, high resolution) of lumen formation of CEACAM1/MCF7 cells untreated, treated with lipofectamine (Lipo control), treated control RNAi or treated with RNAi oligos 2–4 (n= 200, p= 0.001 for RNAi2 vs transfectamine control). Magnification 40x, inset 80x. Acini counted had the following dimensions: D= 40–60-μm, with lumen size D= 8–10 μm.

Figure 5:. Subcellular location of DLK1 is affected by SASH1 expression.

(A) CEACAM1/MCF7 cells before (1) and after treatment (2) with SASH1 RNAi were fractionated by two methods and the proteins analyzed by western blotting for DLK1. In method 1 (left), DLK1 was found mainly in the cytosolic fraction (CE) with an increase in the nuclear fraction (NE). In method 2 (right), three subcellular fractions were analyzed, membrane (MEM), cytoplasm (CE) and nuclear (NE). In this method DLK1 was found mainly in the nuclear fraction with a further increase after treatment with SASH1 RNAi. Markers for the nuclear fraction were HDAC1 and GAPDH for the cytosolic fraction. The degree of contamination of the CE fraction as measured by GAPGH staining was substantial for method 2. (B) Confocal staining of CEACAM1/MCF7 cells with DLK1 (green) reveals substantial perinuclear staining (nuclei counter staied with DAPI, blue). The line drawn across two cells reveals the amount of green and blue staining shown in the histogram below.

Figure 6.. SASH1 expression affects NOTCH1, HES1 and HEY1 expression.

(A) NOTCH1 expression quantitated by qRT-PCR in triplicate from CEACAM1/MCF7 cells treated with control RNAi or SASH1 RNAi. (B) NOTCH1 expression quantitated by qRT-PCR in triplicate from CEACAM1/MCF7 cells transfected with vector control or SASH1-GFP plasmid. (C) HES1 and HEY1 expression quantitated by qRT-PCR in triplicate from CEACAM1/MCF7 cells treated with control RNAi or SASH1 RNAi. (D) HES1 and HEY1 expression quantitated by qRT-PCR in triplicate from CEACAM1/MCF7 cells transfected with vector or SASH1-GFP plasmid.

Figure 7.. SASH1 expression affects protein levels of NOTCH1 and NICD.

(A) CEACAM1/MCF7 cells were transfected with vector or SASH1-GFP plasmid (left) or with RNAi control or SASH1 RNAi (right) and the protein levels of NOTCH1 measured by western blotting of total cell lysates (actin was run as a loading control; numbers refer to relative values for NOTCH1). (B). Same as in (A), except nuclear fractions were prepared and western blotted for NICD with a Lamin A/C control (numbers refer to relative amounts of NICD).

Figure 8.. Treatment of CEACAM1/MCF7 cells with γ-Secretase inhibitor LY411575 inhibits lumen formation.

(A, B) CEACAM1/MCF7 cells were treated with 0.5 μM or 1.0 μM γ-secretase inhibitor LY411575 in 3D culture for 1d or 6d, respectively, and lumen formation scored by microscopic analysis at 6d (n= 200, p= 0.001 for LY411575 treated vs DMSO control). Acini counted had the following dimensions: D= 40–60-μm, with lumen size D= 8–10 μm. (C, D) same as in A and B except cells were treated with vehicle only (0.5% or 1.0% DMSO). (E). Quantitation of lumen from A-D. (F) RNA isolated from 6d acini and expression of NOTCH1 measured by qRT-PCR in triplicate. P values are * <.01, *** <0.001.

Figure 9.. Expression of SASH1, regulated by CEACAM1, leads to branched regulation of DLK1 and NOTCH1.

Genetic analysis of lumen formation of CEACAM1/MCF7 cells reveals SASH1 as a major up-regulated gene that, in turn, up-regulates DLK1 and NOTCH1. Since DLK1 negatively regulates NOTCH1 and its genes (HES1 and HEY1), NOTCH1 signaling is likely under temporal control during lumenogenesis.