Inhibition of protein kinase C delta leads to cellular senescence to induce anti‐tumor effects in colorectal cancer

Abstract Protein kinase C delta (PKCδ) is a multifunctional serine–threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, the molecular mechanism of PKCδ in colorectal cancer (CRC) remains unclear. In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild‐type (wt‐p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt‐p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53‐independent pathway and that PKCδ‐kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence‐like phenotypes, including increased senescence‐associated β‐galactosidase (SA‐β‐gal) staining, low LaminB1 expression, large nucleus size, and senescence‐associated secretory phenotype (SASP) detection. Finally, a kinase inhibitor of PKCδ suppressed senescence‐dependent tumorigenicity in a dose‐dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis. In addition, a novel therapeutic strategy for wt‐p53 CRC is proposed.

Kinase inhibitors for cancer therapy began to be approved about 20 years ago, and more aggressive development of kinase inhibitors has occurred in the last decade. [6][7][8] Kinases are critical factors in intracellular signaling pathways and are implicated in physiological processes such as homeostasis and development, as well as tumor formation, growth, invasion, and metastasis. [9][10][11][12] There are many cases of abnormal cell proliferation due to excessive protein kinase activity in cancer cells, and the development of precise moleculartargeted drugs acting only on specific protein kinases has been actively pursued. 13 Therefore, it is important to identify new protein kinases as target molecules to further understand the molecular pathogenesis of CRC and improve therapeutic outcomes.
Cellular senescence, first reported by Hayflick and Moorhead, is an irreversible cell cycle arrest caused by several factors 14 and is characterized by morphological changes such as hypertrophy and flattening, increased senescence-associated β-galactosidase (SAβ-gal) activity, and senescence-associated secretory phenotype (SASP). 15,16 Cellular senescence is predominant in normal tumor tissues and various precancerous lesions in humans and mice but is decreased in malignant tumors. 17 It is considered a physiological barrier to tumor development and progression. 18,19 Therefore, therapies that induce cellular senescence in tumors have potential as novel therapeutic strategies.
Several studies have shown that genes aberrantly expressed in cancer cells, such as AKAP95 and ZNF768, are involved in the regulation of tumor cell senescence and are potential therapeutic targets. [20][21][22] Additionally, p21 stability and activity are regulated by phosphorylation. 23,24 Thus, kinase activity may influence cellular senescence.
In this study, we identified protein kinase C delta (PKCδ) as a novel therapeutic target kinase for inducing cellular senescence in CRC. We found that PKCδ expression was higher in tumor tissues than in normal tissues of patients with CRC, and PKCδ expression was negatively correlated with p21 expression in p53 wild-type (wt-p53) CRC patients. Furthermore, we demonstrated that inhibition of PKCδ in wt-p53 CRC cell lines induces cellular senescence in a p21-dependent manner and significantly inhibits tumor growth.

| siRNA transfection
Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) was used to achieve transient knockdown with 20 nM siRNA according to the manufacturer's instructions (Table S1).

| Xenograft studies
Seven-week-old female nude mice were obtained from CLEA. The animals were maintained in a pathogen-free animal facility at Jikei University School of Medicine. The mice were randomly assigned to the indicated groups. The cell count was set to 1 × 10 6 , suspended in Matrigel in a total of 100 μL, and injected subcutaneously into the backs of the mice. Tumor size was determined by caliper measurements of the largest (x) and smallest (y) vertical diameters and calculated according to the formula V = π/6 × xy 2 .

| Statistical analysis
Data are presented as the mean ± SD. Statistical significance of differences was evaluated using a two-tailed Student's t-test or one-way ANOVA. The relationship between clinicopathological factors and stainability was analyzed using the χ 2 -test or Fisher's exact test. Differences were considered statistically significant at p < 0.05. Statistical analyses were performed using Prism 8 software (GraphPad, San Diego, CA, USA).

| Supplementary Material and Methods
Details of other material and methods are clarified in Appendix S1.

| Protein kinase C delta is upregulated in human CRC tissues
To explore novel targets of protein kinases against CRC, we categorized serine/threonine kinase activity and examined genes with variable expression in GO analysis. We found 48 serine/threonine kinases upregulated in cancer tissues compared to normal tissues ( Figure 1A,B; Table S2). Specifically, PKCδ showed significantly elevated expression and high reproducibility when compared among its isoforms ( Figure 1C). Therefore, we examined the role of PKCδ in CRC. PKCδ expression was validated by immunostaining using matched pairs of tumors and adjacent normal tissues from patients F I G U R E 1 Protein kinase C delta (PKCδ) is identified as a highly expressed gene among serine-threonine kinases in colorectal cancer (CRC). (A) Representative extraction diagram showing candidate genes identified by RNA sequencing analysis. Gene expression was compared between normal and tumor tissues in the colon of one representative CRC patient with wt-p53. (B) Volcano plots of genes with variable expression in GO analysis categorized by serine/threonine kinase activity. Red plots show increased expression (48 genes) and blue plots show decreased expression (125 genes). (C) mRNA level as fold changes of PKC isoforms was calculated by relative comparison to normal tissue. N, normal tissue; T, tumor tissue. Data are presented as mean ± SD (n = 3). *p = 0.0304. n.s., not significant (paired two-tailed Student's t-test). (D) Normal and tumor tissues from patients with CRC were scored from 1 to 4 by immunohistochemistry and shown in a heatmap (n = 45).  (Table S3). According to the scores of immunostaining degree ( Figure S1), PKCδ expression was significantly higher in cancer tissues than that in normal tissues ( Figure 1D). Additionally, we confirmed the expression of PKCδ at the protein level in various human CRC cell lines ( Figure S2). Bioinformatic analysis of overall survival using the Kaplan-Meier plotter (http:// kmplot.com/analy sis/index.php?p=backg round) showed a relationship between PKCδ and CRC ( Figure S3). These results suggest that PKCδ expression is involved in CRC progression.

| Protein kinase C delta is involved in tumor growth in wt-p53 CRC cell lines
To investigate the biological effects of PKCδ on cancer, we generated PKCδ-KO cells (PKCδ KO-1 and PKCδ KO-2) using the wt-p53 human CRC cell line HCT116 by the CRISPR knockout system. Cell proliferation assays showed that cell growth was significantly inhibited in PKCδ-KO cells compared to that in control cells (Figure 2A). Similar results were obtained from PKCδ-knockdown (KD) cells ( Figure S4A). To investigate the biological impact of PKCδ on CRC, we subcutaneously inoculated HCT116 PKCδ-KO cells into mice. We evaluated tumor size in control and PKC-KO xenografts at necropsy and found that PKC-KO cell-bearing tumors were significantly smaller than control xenografts ( Figure 2J). Similarly, the tumor weight of PKCδ-KO xenografts was significantly lesser than that of the control xenografts ( Figure 2K). Immunohistochemical analysis of xenograft tumors revealed that PKCδ and p21 expression were inversely correlated, and Ki67 expression (a marker for active cell proliferation) in PKCδ-KO xenograft tumors was significantly downregulated compared to the control xenograft tumors ( Figure 2L). These results indicate that PKCδ is involved in the tumor growth of wt-p53 CRC. F I G U R E 2 Protein kinase C delta (PKCδ) is involved in tumor growth in wt-p53 colorectal cancer (CRC) cell lines. (A) Cell proliferation assay of control (parental HCT116) cells and PKCδ-KO (PKCδ KO-1, PKCδ KO-2) cells. Cell proliferation was shown by MTS assay. Data are presented as mean ± SD (n = 4). *p = 0.0021, **p = 0.0007 (one-way ANOVA followed by Bonferroni's multiple comparisons test). (B) Cell proliferation assay of RKO cells treated with control non-targeting siRNA (siControl) or PKCδ-specific siRNA (siPKCδ-1 and siPKCδ-2). Cell proliferation was shown by MTS assay. Data are shown as mean ± SD (n = 4). *p = 0.0041; **p = 0.0004 (one-way ANOVA followed by Bonferroni's multiple comparisons test). (C) Cell proliferation assay of LoVo cells treated with control non-targeting siRNA (siControl) or PKCδ-specific siRNA (siPKCδ-1 and siPKCδ-2). Cell proliferation was measured by MTS assay. Data are shown as mean ± SD (n = 4). *p = 0.0006. n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (D) Cell proliferation assay of DLD-1 cells treated with control non-targeting siRNA (siControl) or PKCδ-specific siRNA (siPKCδ-1 and siPKCδ-2). Cell proliferation was measured by MTS assay. Data are shown as mean ± SD (n = 4). n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (E) Cell proliferation assay of SW480 cells treated with control non-targeting siRNA (siControl) or PKCδ-specific siRNA (siPKCδ-1 and siPKCδ-2). Cell proliferation was measured by MTS assay. Data are shown as mean ± SD (n = 4). n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (F) Cell proliferation assay of SW620 cells treated with control non-targeting siRNA (siControl) or PKCδ-specific siRNA (siPKCδ-1 and siPKCδ-2). Cell proliferation was measured by MTS assay. Data are shown as mean ± SD (n = 4). n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (G) Cell cycle analysis by flow cytometry with propidium iodide (PI) staining in control and PKCδ-KO cells in HCT116. (H) Immunoblot analysis of PKCδ, p21, LaminB1, and GAPDH (loading control) in control and PKCδ-KO cell lysates. Migration of molecular weight markers is shown on the right (kDa). Protein level as fold change of p21 was calculated by comparing protein levels relative to those of control cells after normalization to GAPDH. Data are shown as mean ± SD (n = 3). *p < 0.0001 (one-way ANOVA followed by Bonferroni's multiple comparisons test). (I) Immunoblot analysis of PKCδ, p21, and GAPDH (loading control) in lysates of DLD-1 cells treated with control non-targeting siRNA (siControl) or PKCδ specific siRNA (siPKCδ-1 and siPKCδ-2). Migration of molecular weight markers is shown on the right (kDa). Protein level as fold changes of PKCδ and p21 was calculated by comparing protein levels relative to those of siControl cells after normalization to GAPDH. Data are shown as mean ± SD (n = 3). *p = 0.0027, **p = 0.0052. n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (J) Tumor volumes were measured at indicated time points. Data are shown as mean ± SD (n = 5 per group). *p = 0.0132 (paired two-tailed Student's t-test). (K) Images of tumors at day 21 necropsy of nude mice are shown (left), and tumor mass was measured at that time (right). Data are shown as mean ± SD (n = 5 per group). *p = 0.0025 (paired two-tailed Student's t-test). (L) Representative images of immunohistochemistry staining of PKCδ, p21, and Ki67 in xenograft tumor tissue are shown. Scale bar represents 50 μm. The percentage of cells positive for p21 and PKCδ was calculated from four different magnified fields of view as a percentage of the total number of positive cells in the field of view. Data are shown as mean ± SD (n = 4). *p < 0.0001 (paired two-tailed Student's t-test).

| Suppression of protein kinase C delta expression induces cellular senescence
To verify whether PKCδ-KO cells affect cellular senescence, we   Figure S7). These results suggest that PKCδ phosphorylates p21 (Ser146) and degrades p21 via the ubiquitin-proteasome system. They also show that suppression of PKCδ expression in wt-p53 HCT116 inhibits p21 (Ser146) phosphorylation and prevents p21 degradation, which allows p21 accumulation and cell cycle arrest, as well as cellular senescence ( Figure 4H).

F I G U R E 4
Protein kinase C delta (PKCδ) phosphorylates p21 independent of p53 and degrades p21 in the ubiquitin-proteasome system. (A) Control cells and PKCδ-knockout (KO) cells transformed with control non-targeting siRNA (siControl) or p53-specific siRNA (sip53-1 and sip53-2) were lysed and analyzed for PKCδ, p53, p21, and GAPDH (loading control) by immunoblotting. Migration of molecular weight markers is shown on the right (kDa). (B) No-treatment HCT116 p53(−/−) cells or HCT116 p53(−/−) cells transformed with control nontargeting siRNA (siControl) or PKCδ-specific siRNA (siPKCδ-1 and siPKCδ-2) were lysed and analyzed for PKCδ, p53, p21, and GAPDH (loading control) by immunoblotting. Parental HCT116 cells were also used as positive controls. Migration of molecular weight markers is shown on the right (kDa). (C) Control and PKCδ-KO cells and GFP-PKCδ transfected PKCδ-KO cells were lysed and subjected to immunoblot analysis for PKCδ, p21, p53, and GAPDH (loading control). Migration of molecular weight markers is shown on the right (kDa). (D) mRNA expression of p53 and p21 in control and PKCδ-KO cells was measured by qPCR. HPRT1 was used as an internal standard, and fold change was calculated by comparing mRNA expression levels relative to those of control cells. Data are shown as mean ± SD (n = 6). n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (E) Immunoblot analysis of phospho-p21 (Ser146), phospho-p21 (Thr145), p21, PKCδ, and GAPDH (loading control) in lysates from control and PKCδ-KO cells. Migration of molecular weight markers is shown on the right (kDa). Protein levels of p-p21(Ser146) and p21 were normalized with those of GAPDH, and the amount of change was calculated by comparing p-p21 (Ser146)/p21 in PKCδ-KO cells relative to that in control cells. Data are shown as mean ± SD (n = 3). *p = 0.0014; **p = 0.0007 (one-way ANOVA followed by Bonferroni's multiple comparisons test). (F) Control and PKCδ-KO cells transfected with GFP-PKCδ were lysed and immunoblot analyzed for phospho-p21 (Ser146), p21, PKCδ, and GAPDH (loading control). Migration of molecular weight markers is shown on the right (kDa). (G) Immunoprecipitation analysis of control and PKCδ-KO cells transfected with HA-tagged ubiquitin. Accumulation of polyubiquitinated proteins was induced by MG132 treatment (5 μM, 5 h) prior to cell lysis. Cells were analyzed by immunoprecipitation and immunoblotting using the antibodies shown. Migration of molecular weight markers is shown on the right (kDa). (H) Inhibition of genetic targeting of PKCδ in HCT116 colorectal cancer (CRC) cells (wt-p53) causes not only cell cycle arrest but also induction of cell senescence and even secretion of SASP, resulting in cell growth inhibition.

| VTX-27 suppresses tumorigenesis in a p21-dependent way
To investigate the biological effects of PKCδ inhibitors on senescence in CRC cells, we used PKCδ inhibitors. Among various PKCδ inhibitors, including rottlerin, we selected VTX-27, an inhibitor particularly selective for PKCδ. 26 We confirmed a concentrationdependent decrease in phospho-PKCδ (Thr505) by VTX-27 at the protein level, a decrease in phospho-p21 (Ser146) expression, and an increase in p21 expression ( Figure 5A). We evaluated the IC50 in HCT116 cells ( Figure S8A) and demonstrated a concentrationdependent decrease in the proliferative potential of VTX-27 in the proliferation assay ( Figure 5B). To investigate this phenomenon in an environment similar to the in vivo environment, we performed spheroid formation assays. A significant concentration-dependent reduction in spheroid size was observed at concentrations with VTX-27 above 20 nM (Figures 5C and S8B). Cell count and luminescence measurements also confirmed the effect of this inhibitor on tumor formation ( Figure 5D,E). We performed apoptosis assays to assess the effect of VTX-27 on cell death on HCT116 cells but found no apparent apoptosis ( Figure S8C). We subsequently performed immunofluorescence staining of spheroids. We confirmed decreased expression of phospho-PKCδ (Thr505) by VTX-27 and increased expression of p21 and decreased expression of LaminB1 ( Figure 5F). We also revealed nuclear enlargement in cells treated with VTX-27 ( Figure 5G). Contrastingly, no change in p53 expression was observed ( Figure S9A). The decrease in Ki67 expression by VTX-27 was also indicative of decreased proliferative capacity ( Figure S9B). These data demonstrate that the suppression of PKCδ activity in a near-in vivo environment induces p53-independent cellular senescence and leads to antitumor effects.

| Protein kinase C delta in human wt-p53 colorectal cancer tissues correlates positively with cancer progression and negatively with p21 expression
The incidence of p53 mutations varies depending on the site of tumor development in the colon, and their frequency has been reported to be higher in left-sided (distal) CRC than in right-sided (proximal) CRC. 27,28 To collect cases of wt-p53 in human cancer tissue, 71 cases of right-sided colon cancer were randomly selected and their p53 status was determined by immunostaining patterns (Figure S10A; refs. [29]). Of the 71 cases, 32 had wt-p53 CRC and 37 had mt-p53 CRC. Interestingly, clinical stage and T classification were positively correlated with staining intensity in wt-p53 ( Figure 6A; Table S4). However, there was no obvious difference in mt-p53 ( Figure 6A; Table S5). These results suggest that PKCδ functions in a tumor-promoting manner in wt-p53 CRC.
Based on these findings, we evaluated p21 expression in wt-p53 CRC. Intriguingly, the weaker the staining of PKCδ, the higher the expression of p21; the stronger the staining of PKCδ, the lower the expression of p21 (Figures 6B and S10B). These results suggest that the positive correlation between PKCδ expression and cancer progression in wt-p53 CRC is due to the abrogation of G1 phase arrest mediated by p21. Data from human tissues are consistent with those from in vitro experiments.

| DISCUSS ION
We concentrated on the PKC family, which functions differently in different types of cancer 30 and PKCδ, which was significantly upregulated. Intriguingly, some reports show the low expression of PKCδ, while recent reports show its high expression, indicating controversial findings on PKCδ expression in CRC. [31][32][33][34] Additionally, we confirmed that PKCδ expression was significantly elevated in CRC tissues based on immunostaining analysis.
In functional analysis of PKCδ, PKCδ inhibition increased p21 protein levels. p21 is a key player in the PKCδ-dependent cellular senescence. Interestingly, this phenomenon appears to be p53independent. Pharmacological inhibition of PKCδ in wt-p53 cell lines increased p21 protein levels in a p53-independent manner, whereas restoring PKCδ function reversed the increase in p21 protein levels. This mechanism was supported by the result of the suppression of phosphorylation of p21 by PKCδ, which suppressed ubiquitinproteasomal degradation of p21 and resulted in p21 accumulation.
Although direct involvement of p21 in PKCδ-mediated biological effects has been reported, 35 its role in cellular senescence in CRC F I G U R E 5 VTX-27 suppresses tumorigenesis in a p21-dependent way. (A) Immunoblot analysis of phospho-PKCδ (Thr505), phospho-p21 (ser146), p21, and GAPDH (loading control) in HCT116 colorectal cancer (CRC) cell lysates treated with DMSO or VTX-27 for 48 hours. Migration of molecular weight markers is shown on the right (kDa). (B) Cell proliferation assay of HCT116 cells treated with DMSO or VTX-27. Cell proliferation was measured by the MTS assay. Data are shown as mean ± SD (n = 3). *p = 0.0015; **p < 0.0001. n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (C) three-dimensional multicellular spheroid formation after 7 days of culture of HCT116 cells on ultra-low adhesion plates and treatment with DMSO or VTX-27. Scale bar indicates 50 μm. (D) The number of spheroids larger than 40 μm per well was counted. Data are shown as mean ± SD (n = 4). *, p < 0.0001. n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (E) Cell viability of spheroids was measured by ATP luminescence. The 3D Cell Viability Assay was used to measure cell viability. Data are presented as mean ± SD (n = 4). *p < 0.0345; **p < 0.0001. n.s., not significant (one-way ANOVA followed by Bonferroni's multiple comparisons test). (F) Spheroids formed by treatment with DMSO or VTX-27200 nM were immunofluorescently stained with anti-phospho-PKCδ (Thr505), anti-p21, anti-LaminB1, anti-p53, and anti-Ki67 antibodies (green). Nuclei were stained with DAPI (blue). Scale bar indicates 50 μm. (G) The area of nuclei of control (DMSO) cells and cells treated with VTX-27 was measured. Data are presented as mean ± SD (n = 100). *p < 0.0001 (paired two-tailed Student's t-test).

F I G U R E 6
In wt-p53 colorectal cancer, protein kinase C delta (PKCδ) expression is positively and negatively correlated with cancer progression and p21 expression, respectively. (A) Tumor tissues from colorectal cancer patients were immunohistochemically stained with anti-PKCδ antibody, divided into high and low staining, and the correlation between stage classification and staining intensity and T classification and staining intensity were plotted (wt-p53; n = 32, mt-p53; n = 39). (B) Immunohistochemical staining of wt-p53 colorectal tumor tissue with anti-PKCδ and anti-p21 antibodies. Representative samples with positive and negative p21 expression are presented. Scale bar indicates 100 μm. The percentage of cells positive for p21 is the ratio of the number of positive cells to the total number of cells in the field of view and was calculated from four different magnified fields of view. Data are shown as mean ± SD (n = 6). *p < 0.0001 (paired two-tailed Student's t-test).
has never been elucidated. Our findings show that PKCδ induces cellular senescence through its involvement in phosphorylation of p21, which is the first report to establish direct regulation of p21 by PKCδ. Cellular senescence, defined as a state of cell cycle arrest, has a potent inhibitory effect on tumorigenesis. Thus, molecules that promote senescence have long been expected to be used as anticancer agents. 19,36 In this study, we showed that PKCδ plays an important role in cellular senescence in wt-p53 CRC. In the CRC cell line HCT116, inhibition of PKCδ induced marked cellular senescence, resulting in the production of several cytokines involved in SASP.
This finding provides evidence that PKCδ-dependent cellular senescence is induced by cyclin-dependent kinase inhibitor p21-mediated senescence.
Protein kinase C delta, a PKC isoform, activates p21 in a p53dependent manner in response to DNA damage and cell cycle. [37][38][39][40] Hence, PKCδ functions as a tumor suppressor. Conversely, high PKCδ expression has been correlated with poor prognosis, and PKCδ has been reported to have tumor-promoting functions in liver and breast cancers. [41][42][43] PKCδ is known to be associated with anticancer drug resistance in lung cancer. 44 In this study, PKCδ was highly expressed in CRCs. We also found that it suppresses senescence in wt-p53 CRC cells and contributes to tumor enhancement. This suggests that PKCδ may function as a tumor-promoting factor in CRC, whereas PKCδ functions as a tumor suppressor in precancerous lesions, which are susceptible to DNA damage and may function progressively against cancer after cancer transformation.
Cellular senescence involves SASPs that secrete cytokines and chemokines, which act in an autocrine manner and enhance cellular senescence by CXCR2 and its ligand factors. 45 In contrast, it has been shown to act on the surrounding cells in a paracrine manner.
However, the paracrine action of SASP not only enhances cellular senescence but also has negative effects, many of which have undesirable effects on the organism, such as carcinogenesis and inflammation. 46 In the present study, we observed a marked increase in the SASP factors IL-6, IL-8, and TNFα in the medium of PKCδ-KO HCT116 cells. This phenomenon is consistent with cellular senescence; however, whether this is an autocrine enhancement of CXCR2-mediated senescence or a paracrine enhancement of cancer-promoting effects is a subject for future studies on SASP factor inhibition. If SASP secretion is associated with PKCδ inhibition, it may affect carcinogenesis and cancer progression in a paracrine manner. We believe that the combination of PKCδ inhibition with anti-chemokine antibodies may have further anti-tumor effects.
Although cellular senescence is regulated by the p53 and p16-pRb pathways, several studies have shown that this process is cell type-specific and can be regulated by different senescence pathways independent of p53 and p16. 47,48 In the present study, p16 did not appear to be involved in PKCδ-induced cellular senescence because suppression of PKCδ expression in HCT116 cells induced p21 and SAβ-gal expression regardless of p16 expression status ( Figure S4F). Contrastingly, suppression of PKCδ expression upregulated pRb expression, implying that Rb gene activation is regulated by a p21-dependent pathway rather than the p16-pRb pathway, which requires further clarification.
We found that PKCδ was upregulated in CRC tissues regardless of p53 status. Additionally, a positive correlation was found between PKCδ and tumor progression only in wt-p53 CRC. Although we could not find a relationship between PKCδ and cellular senescence in mt-p53 CRC cell lines in the present study, the expression of PKCδ was upregulated in mt-p53 CRC from an early stage, and the involvement should be investigated from a tumorigenesis perspective in a future study. Finally, we propose that PKCδ is a promising biomarker and therapeutic target for colorectal cancer in wt-p53 CRC.

ACK N OWLED G M ENTS
We would like to thank Dr Katsuhiko Aoki and Naoko Tago for technical support. We would like to thank Editage (www.edita ge.com) for English language editing.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The unprocessed source data and the statistical source data that support the findings of this study are available, and correspondence and requests for materials should be addressed to kyoshida@jikei. ac.jp.

E TH I C S A PPROVA L S TATE M E NT
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Review