NOTCH signaling is required for formation and self-renewal of tumor-initiating cells and for repression of secretory cell differentiation in colon cancer

Abstract

NOTCH signaling is critical for specifying the intestinal epithelial cell lineage and for initiating colorectal adenomas and colorectal cancers (CRC). Based on evidence that NOTCH is important for the maintenance and self-renewal of cancer-initiating cells in other malignancies, we studied the role of NOTCH signaling in colon cancer-initiating cells (CCIC). Tumors formed by CCICs maintain many properties of the primary CRCs from which they were derived, such as glandular organization, cell polarity, gap junctions, and expression of characteristic CRC molecular markers. Furthermore, CCICs have the property of self-renewal. In this study, we show that NOTCH signaling is 10- to 30-fold higher in CCIC compared with widely used colon cancer cell lines. Using small-molecule inhibition and short hairpin RNA knockdown, we show that NOTCH prevents CCIC apoptosis through repression of cell cycle kinase inhibitor p27 and transcription factor ATOH1. NOTCH is also critical to intrinsic maintenance of CCIC self-renewal and the repression of secretory cell lineage differentiation genes such as MUC2. Our findings describe a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest that inhibition of NOTCH signaling may improve CRC chemoprevention and chemotherapy.

Figure 1. Colon cancer initiating cells maintain histopathological properties of CRC in vitro

(A) H&E, CEA, CK20 and CK7 staining in tumors generated by injection of CCIC in flank of NOD/SCID mice. One representative out of 8 tumors is shown (B) Flow cytometry analysis for CD133, CD44, ESA and ALDH1 on CCIC cultured in non-adherent culture conditions. Expression of these markers is conserved on long term culture in vitro. (C) H&E staining of xenografts generated in mice from CCIC cultured in vitro for indicated times showing that CCIC can maintain glandular differentiation and histologically similar tumors after long term culture. (D) H&E, CK20, β-catenin and CK7 staining of 3D cultures of single CCIC plated and cultured for 3 weeks showing CCIC can generate tumor foci in vitro.

Figure 2. NOTCH signaling is high in CCIC cultures

Expression of NOTCH components JAG1, NOTCH1 and target HES1 in CCIC is higher as compared to normal colon crypts.

Figure 3. NOTCH signaling is active in CCIC

(A) FACS analyses of CCIC-1 with NOTCH/ RBPJκ GFP reporter. 33% of have high levels of GFP as compared to control population without reporter. Histogram of CCIC alone, CCIC with reporter and CCIC with reporter treated with DAPT 10μM for 24h. NOTCH inhibition in CCIC decreases expression of GFP from the reporter. (B) Immunofluorescence for NICD in 3D cultures. Tumor foci formed from CCIC express cytoplasmic CK20 (green) and nuclear NICD (red). Sections were counterstained with DAPI (blue). Merged image shows that NICD localizes to the nucleus of tumor foci.

Figure 4. DAPT blocks NOTCH signaling in CCIC and causes expression of goblet cell markers

(A) Expression of HES1 and HES5 in CCIC decreases on treatment with DAPT (left). DAPT treatment inhibits cleavage of NOTCH by γ-secretase as NICD is not detected by immunoblotting in CCIC (right). (B) DAPT relieves repression caused by expression of HES1 on MATH1, MUC2 and CDK inhibitor p27. (C) MUC2 + cells is increased by 2.5 fold on transient treatment with DAPT. Counted MUC2+ cells in 100 colonies in 5 independent experiments.

Figure 5. DAPT inhibits formation of tumor foci and causes apoptosis in CCIC.

(A) Single cells plated in 3D culture and treated with 10μM DAPT continuously are unable to initiate to tumor foci as compared to control. (B) Cell cycle analysis of cells treated DAPT shows increase in sub-G1 population indicating apoptosis. (C) CCIC treated with DAPT express cleaved caspase-3, show decrease in HES1 and absence of NICD consistent with apoptosis.

Figure 6. RBPJκ knockdown causes expression of goblet cell markers and apoptosis in CCIC

(A) Lentiviral infection of shRNA against RBPJκ suppresses expression of RBPJκ mRNA and causes downregulation of NOTCH target HES1 (left). shRNA knockdown decreases RBPJκ protein levels (middle). Knockdown of RBPJκ increases expression of secretory lineage markers MATH1, MUC2, decreases enterocyte marker CK20 and increases expression of CDK inhibitor p27 compared to control (right). (B) CCIC infected with scrambled or shRNA for RBPJκ and plated in 3D culture for 3 weeks. CCIC expressing RBPJκ shRNA cannot initiate tumor foci in 3D culture. (C) Cell cycle analysis of cells expressing RBPJκ shRNA shows gradual increase in sub-G1 peak post infection indicating apoptosis. (D) Apoptosis in CCIC by RBPJκ shRNA is confirmed by increase in cleaved caspase-3.