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. 2018 Mar;37(12):1669-1684.
doi: 10.1038/s41388-017-0060-8. Epub 2018 Jan 18.

Mutant p53 Gain of Function Underlies High Expression Levels of Colorectal Cancer Stem Cells Markers

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Free PMC article

Mutant p53 Gain of Function Underlies High Expression Levels of Colorectal Cancer Stem Cells Markers

Hilla Solomon et al. Oncogene. .
Free PMC article

Abstract

Emerging notion in carcinogenesis ascribes tumor initiation and aggressiveness to cancer stem cells (CSCs). Specifically, colorectal cancer (CRC) development was shown to be compatible with CSCs hypothesis. Mutations in p53 are highly frequent in CRC, and are known to facilitate tumor development and aggressiveness. Yet, the link between mutant p53 and colorectal CSCs is not well-established. In the present study, we set to examine whether oncogenic mutant p53 proteins may augment colorectal CSCs phenotype. By genetic manipulation of mutant p53 in several cellular systems, we demonstrated that mutant p53 enhances colorectal tumorigenesis. Moreover, mutant p53-expressing cell lines harbor larger sub-populations of cells highly expressing the known colorectal CSCs markers: CD44, Lgr5, and ALDH. This elevated expression is mediated by mutant p53 binding to CD44, Lgr5, and ALDH1A1 promoter sequences. Furthermore, ALDH1 was found to be involved in mutant p53-dependent chemotherapy resistance. Finally, analysis of ALDH1 and CD44 in human CRC biopsies indicated a positive correlation between their expression and the presence of oncogenic p53 missense mutations. These findings suggest novel insights pertaining the mechanism by which mutant p53 enhances CRC development, which involves the expansion of CSCs sub-populations within CRC tumors, and underscore the importance of targeting these sub-populations for CRC therapy.

Conflict of interest statement

Compliance with ethical standards

Conflict of interest The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Mutant p53 gain of function endows colorectal cancer cell lines with higher oncogenic potential. The colorectal adenocarcinoma-derived cell line, SW480 that endogenously expresses mutant p53R273H,P309S was stably infected with shRNA against p53 (shp53) or with shRNA against nonspecific sequence (shCon), as a control, a Western Blot displays mutant p53 protein levels. GAPDH serves as loading control. This figure combines two independent detections of p53 and GAPDH of the same gel. The two original images are presented in Supplementary Figure 1. b The shCon and shp53 SW480 cell lines (2 × 105 or 1 × 104) were injected into left and right limb of nude mice, respectively, followed by tumor growth surveillance. After 8–10 weeks mice were sacrificed and tumors size was measured. Asterisks denotes statistical significance (p < 0.01, n = 6). c Three independent newly infected shCon and shp53 SW480 cell lines were mixed and were injected (7 × 105) into left and right limb of nude mice, respectively, followed by tumor growth surveillance. After 5 weeks mice were sacrificed and tumors weight was measured. Asterisks denotes statistical significance (p < 0.05, n = 5). d Photos of tumors presented in c that illustrate the significant differences in the size of tumors obtained from injection of shCon and shp53 SW480 cells, e The established SW480 cell lines were treated with 5-FU (50 μΜ) for 72 h, followed by AnnexinV staining and ImageStream X analysis (Materials and methods). Graph in left panel indicates on percentage of apoptotic cells upon 5-FU treatment, normalized to NT cells. Right panel is a representative photo of an apoptotic cell, f RKO isogenic cell lines that express either WTp53 (RKO+/+) or mutant p53R248W (RKO+/m), or knocked-out for p53 (RKO−/−) [20] were treated with cisplatin (2.5 μg/ml) for 72 h, followed by AnnexinV staining and ImageStream X analysis. Graph in left panel indicates on percentage of apoptotic cells upon cisplatin treatment, normalized to NT cells. Right panel is a representative photo of an apoptotic cell. Graphs represent an average of three experiments. Error bars represent SE. Asterisk denotes statistical significance. For more representative photos obtained by ImageStream X see Supplementary Figure 13 A–B
Fig. 2
Fig. 2
Mutant p53-expressing cells contain larger CD44Br and Lgr5Br sub-populations a, b RKO isogenic cell lines were immuno-stained with anti-Lgr5 antibody and the size of Lgr5Br sub-population was measured by ImageStream X. a Graph presenting an averaged percentage of Lgr5Br cells obtained from three independent experiments. Error bars represent SE. b Representative photo of Lgr5Br cedi, c Organoids were produced from intestinal epithelial cells extracted from WTp53, p53 knock-out (p53 KO), or WTp53/mutant p53R172H heterozygous mice, as previously described [15]. Lgr5 mRNA expression levels were measured by qRT-FCR using specific primers. Graph represents an average of three independent organoids pools. Error bars represent SE. d RKO isogenic cell lines were immuno-stained with either anti-CD44 antibody that recognizes all CD44 isoforms and the size of CD44Br sub-populations was measured by FACS. Left: Graph presenting an averaged percentage of CD44Br cells obtained from three experiments. Error bars represent SE. Right panel shows representative dot plots indicating on percentage of CD44Br sub-populations, e-g The established shCon and shp53 SW480 cells were immuno-stained with either anti-CD44 antibody that recognizes all CD44 isoforms and the size of CD44Br sub-populations was measured by ImageStream X. e Graph presenting an averaged percentage of cells obtained from four experiments, f Representative plot of the mean pixel intensity of membranal CD44 in shCon and shp53 cell populations, g Representative photo of CD44Br cell obtained by ImageStream X. For more representative photos obtained by Image-Stream X see Supplementary Figure 13 C–D. h mRNA expression levels of CD44 in the established SW480 tumors were measured by qRT-PCR using specific primers. Asterisk denotes statistical significance
Fig. 3
Fig. 3
Mutant p53 induces larger ALDHBr sub-populations. The RKO isogenic cell lines (a) and established shCon and shp53 SW480 cells (b) were subjected to ALDH activity assay and the size of ALDHBr sub-populations was measured by FACS. Left panels show dot plots of representative experiment indicating on percentage of ALDHBr cells. Right panels show graphs of averaged percentage of ALDHBr cells obtained from three experiments. Error bars represent SE. c SW480 cell line endogenously expressing mutant p53R273H,p309S was sorted by FACS according to ALDH activity levels. Then, the sorted populations were incubated in suspension under serum free spheroids promoting media. Following 2 weeks spheroids were counted via microscopic examination. Graph represents an averaged number of spheres detected in three experiments. Right panels are photos of representative fields, d-f mRNA expression levels of ALDH1A3 in RKO cell lines (d), ALDH1A1 in SW480 cell lines (e), and ALDH1A1 in the established SW480 tumors (f) were measured by qRT-PCR using specific primers, g-i MEFs that were extracted from wild-type p53 mice (+/+), p53 KO mice (−/−), and mutant p53R172H knock-in mice (m/m) were subjected to standard reprogramming protocol [31] followed by measurement of mRNA expression levels of Nanog g, ALDH1A1 h and ALDH3A1 i by qRT-PCR using specific primers, j ChIP analysis of SW480 cells. Endogenous mutant p53 protein was immunoprecipitated using p53-specific antibody (anti-p53 Ab). Empty beads were used as a negative control (Beads only). qRT-PCR was performed using specific primers directed to ALDH1A1, CD44 and Lgr5 promoters. Values were normalized to 1% input. Results are average of three experiments. Error bars represent SE. Asterisk denotes statistical significance
Fig. 4
Fig. 4
Mutant p53-dependent chemotherapy resistance is mediated by ALDH. a, b the established shCon and shp53 SW480 and the RKO isogenic cell lines were treated with cisplatin (2.5 μg/ml) for 72 h. mRNA levels of ALDH1A1 a and ALDH1A3 b were measured in SW480 and RKO, respectively, by qRT-PCR using specific primers, c, d The established shCon and shp53 SW480-expressing mutant p53R273H,F309S were transiently transfected with pcDNA3-HA-ADH to over-express ALDH1A1. Following 24 h, cells were treated with cisplatin (2.5 μg/ml) for additional 72 h. Then, cells were collected and subjected to AnnexinV staining and to ImageStream X analysis. Right panel indicates on percentage of dead cells. c mRNA levels of ALDH1A1 indicating on successful transfection, resulting in ALDH1A1 over-expression in the cells. d Graph indicating on percentage of apoptotic cells upon cisplatin treatment, normalized to NT cells. All graphs represent an average of three experiments. Error bars represent SE. Asterisk denotes statistical significance. OE over-expression. For more representative photos obtained by ImageStream X see Supplementary Figure 13E
Fig. 5
Fig. 5
Mutant p53 correlates with elevated ALDH levels in patients with colorectal carcinoma. Tumor biopsies obtained from sporadic colorectal carcinoma patients were immuno-stained for ALDH and p53, and underwent TP53 sequencing, a Diagrammatic presentation of the status of ALDHA1 along with the TP53 sequencing data for every individual case. Π labeling index, b Representative photos of ALDH and p53 staining showing increased ALDH levels in a mutant p53 case compared to a WTp53 one. Arrows denote positive ALDH staining. Scale bar: 100 μm. c Cases with missense mutations of p53 exhibit increased ALDH levels vs. WTp53 + indel ones. Asterisk denotes statistical significance (p-value < 0.05)

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