p15INK4B is an alternative marker of senescent tumor cells in colorectal cancer

Abstract

Senescent tumor cells are nonproliferating tumor cells which are closely related to cancer progression by secreting senescence-related molecules, called senescence-associated secreting phenotypes. Therefore, the presence of senescent tumor cells is considered a prognostic factor in various cancer types. Although senescence-associated β-galactosidase staining is considered the best marker for detection of senescent tumor cells, it can only be performed in fresh-frozen tissues. p16^INK4A, a cyclin-dependent inhibitor, has been used as an alternative marker to detect senescent tumor cells in formalin-fixed paraffin-embedded tissues. However, other reliable markers to detect senescent tumor cells is still lacking. In the present study, using public single-cell RNA-sequencing data, we found that p15^INK4B, a cyclin-dependent kinase inhibitor, is a novel marker for detection of senescent tumor cells. Moreover, p15^INK4B expression was positively correlated with that of p16^INK4A in colorectal cancer tissues. In in vitro studies, mRNA expression of p15^INK4B was increased together with that of p16^INK4A in H₂O₂- and therapy-induced cancer senescence models. However, the mRNA level of p15^INK4B did not increase in the oncogene-induced senescence model in primary colonic epithelial cells. In conclusion, p15^INK4B is a potential alternative marker for detection of senescent tumor cells together with conventional markers in advanced stages of colorectal cancer.

Keywords: CDK, cyclin dependent kinase; CRC, colorectal cancer; Cellular senescence; Colorectal cancer; FBS, fetal bovine serum; FFPE, formalin-fixed paraffin-embedded; GSEA, gene set enrichent analysis; H3K9me3, histone H3 lysine 9 trimethylation; IHC, immunohistochemistry; SA-β-Gal, senescence-associated β-galactosidase; STC, senescent tumor cell; Senescence marker; Senescent tumor cells; p15INK4B; p16INK4A; scRNA-seq, single cell RNA sequencing.

Fig. 1

Conventional senescence markers for detection of STCs in CRC. (A) A surgically removed fresh CRC tissue was cut into two segments. One segment was frozen and used for SA-β-Gal staining, and the other segment was processed for FFPE to perform IHC analysis. The nearest sections from frozen tissue (for SA-β-Gal staining, left) and FFPE tissue (for IHC p16^INK4A staining, right) were used for analysis. Arrows indicate SA-β-Gal positive cells. (B) IHC analysis with the conventional senescence markers p21^Waf1, p27^Kip1, and p53, is shown. (C) Serial sections of CRC tissue were stained with p16^INK4A and H3K9me3. Arrows indicate immune-like cells which are positive for p21^Waf1or p53.

Fig. 2

p15^INK4Bis an alternative marker for detection of STCs in CRC in vivo. (A) Clusters of EPCAM-positive cancer cells from patient #8 and feature plots of MKI67 and CDKN2A.(B) Dot plot showing the expression pattern of the senescence marker CDKN2A and the proliferation marker MKI67 in each cluster. (C) GSEA using a cellular senescence-related gene set. Leading edges which are upregulated in cluster 1 are shown in the box below. (D) Dot plot showing the expression pattern of CDKN2A and CDKN2B in each cluster. (E) IHC analysis using serial sections from p16^INK4A-positive FFPE CRC tissue. (F) IHC analysis using serial sections from p16^INK4A-negative FFPE CRC tissue. (G) Table showing correlations between p15^INK4B and p16^INK4A expression in FFPE IHC samples. The p value was calculated using Fisher's exact test.

Fig. 3

STC p15^INK4Bexpression in vitro. (A) SA-β-Gal staining in two cancer cell senescence models, H₂O₂-induced (60 μM) and doxorubicin (Dox)-induced (50 nM) senescence models (n = 4, each). The p values are obtained using Mann-Whitney U test. (B) DAPI staining revealed heterochromatin foci, in both H₂O₂-induced (60 μM) and doxorubicin (Dox)-induced (50 nM) senescence models. The proportion of SAHF-positive cells out of total cells was shown in the bar graph and p values were obtained from chi-square test. (C) mRNA expression of CDKN2A and CDKN2B in H₂O₂-induced (60 μM) and doxorubicin (Dox)-induced (50 nM) senescence models (n = 3, each). The p values were obtained using Student's t-test.