Skin epidermis lacking the c-Myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene

The target gene(s) required for Myc-mediated tumorigenesis are still elusive. Here we show that while endogenous c-Myc is surprisingly dispensable for skin homeostasis and TPA-induced hyperplasia, c-Myc-deficient epidermis is resistant to Ras-mediated DMBA/TPAinduced tumorigenesis. This is mechanistically linked to p21(Cip1), which is induced in tumors by the activated Ras-ERK pathway but repressed by c-Myc. Acute elimination of c-Myc in established tumors leads to the up-regulation of p21(Cip1), and epidermis lacking both p21(Cip1) and c-Myc reacquires normal sensitivity to DMBA/TPA-induced tumorigenesis. This identifies c-Myc-mediated repression of p21(Cip1) as a key step for Ras-driven epidermal tumorigenesis.     

p62/SQSTM1 synergizes with autophagy for tumor growth in vivo

Autophagy is crucial for cellular homeostasis and plays important roles in tumorigenesis. FIP200 (FAK family-interacting protein of 200 kDa) is an essential autophagy gene required for autophagy induction, functioning in the ULK1–ATG13–FIP200 complex. Our previous studies showed that conditional knockout of FIP200 significantly suppressed mammary tumorigenesis, which was accompanied by accumulation of p62 in tumor cells. However, it is not clear whether FIP200 is also required for maintaining tumor growth and how the increased p62 level affects the growth in autophagy-deficient FIP200-null tumors in vivo. Here, we describe a new system to delete FIP200 in transformed mouse embryonic fibroblasts as well as mammary tumor cells following their transplantation and show that ablation of FIP200 significantly reduced growth of established tumors in vivo. Using similar strategies, we further showed that either p62 knockdown or p62 deficiency in established FIP200-null tumors dramatically impaired tumor growth. The stimulation of tumor growth by p62 accumulation in FIP200-null tumors is associated with the up-regulated activation of the NF-κB pathway by p62. Last, we showed that overexpression of the autophagy master regulator TFEB^S142A increased the growth of established tumors, which correlated with the increased autophagy of the tumor cells. Together, our studies demonstrate that p62 and autophagy synergize to promote tumor growth, suggesting that inhibition of both pathways could be more effective than targeting either alone for cancer therapy.     

MTS1/p16/CDKN2 lesions in primary glioblastoma multiforme.

The multiple tumor suppressor 1 (MTS1) gene encoding the p16 inhibitor of cyclin-dependent kinase 4 is deleted or mutated in a wide variety of human tumor cell lines, but the importance of this gene as a tumor suppressor in vivo appears to be highly dependent on tumor type. Because MTS1/p16/CDKN2 and the homologous MTS2/p15 gene map to a region of chromosome 9p21, which is frequently deleted in malignant gliomas, we searched for lesions of these genes in primary biopsies of glioblastoma multiforme (GBM). Our analysis confirms a sizable frequency of homozygous deletion of MTS1/p16/CDKN2 (9/27 cases) and also reveals a low but detectable frequency of intragenic DNA lesions (one point mutation in exon 2 leading to premature termination) among GBMs that retain one or both copies of the gene. No mutations were found in exon 2 of MTS2/p15 (12 cases examined), and one GBM showed a DNA deletion breakpoint in the 30 kb between MTS1/p16/CDKN2 and MTS2/p15 resulting in deletion of MTS1/p16/CDKN2 with retention of MTS2/p15. In contrast to the high-grade tumors, none of 12 low-grade gliomas showed MTS1/p16/CDKN2 deletions. These data support a role for MTS1/p16/CDKN2 as a tumor suppressor gene in the in vivo evolution of GBMs. Given that two tumors with hemizygous MTS1/p16/CDKN2 deletions and loss of heterozygosity for chromosome 9p21 did not contain detectable intragenic mutations, there may be one or more additional relevant 9p21 tumor suppressor genes.     

Involvement of p21 (waf1) in merlin deficient sporadic vestibular schwannomas

Previous studies have demonstrated that merlin acts as a tumor suppressor by blocking Ras-mediated signaling. However, the mechanism by which merlin controls cell proliferation has remained obscure. Here we show that merlin deficient tumors exhibited loss of p21, concomitant with elevated CDKs/cyclin D1 levels in sporadic vestibular schwannomas (VS) from clinic patients. Likewise, silencing of merlin gene expression in the cell lines resulted in down-regulation of p21. Furthermore, we find that merlin-enhanced p21 protein stability, rather than increased RNA accumulation, was responsible for the elevated p21 levels. Interestingly, p21 was required to maintain merlin levels and the inhibitory effect of merlin on Ras signaling was partially overridden by knockdown of p21. Consistent with the observation that over-expression of merlin arrested cell growth at G1-phase, the current study indicates that merlin exerts its antiproliferative effect, at least in part, by maintaining p21 expression, and loss of p21 is a prominent feature of merlin deficient schwannomas.     

胃癌组织cyclinE和p21~(WAF1/CIP1)蛋白表达的意义

目的 :探讨cyclinE和p2 1WAF1/CIP1蛋白在胃癌发生发展中的作用及其表达的意义。方法 :采用免疫组化S P法检测正常胃黏膜、萎缩性胃炎伴肠上皮化生、萎缩性胃炎伴不典型增生各 2 0例和 78例胃腺癌组织中cyclinE和 p2 1WAF1/CIP1蛋白表达。结果 :cyclinE蛋白阳性表达在胃癌组高于正常胃黏膜、萎缩性胃炎伴肠上皮化生组 ,而 p2 1WAF1/CIP1蛋白表达则相反 ,差异均有显著性 (P <0 0 5 ) ;cyclinE、p2 1WAF1/CIP1蛋白表达与胃癌细胞分化程度相关 (P <0 0 5 ) ;有肝转移的胃癌组cyclinE阳性表达率高于无肝转移组 (P <0 0 5 ) ;有淋巴结转移组 p2 1WAF1/CIP1蛋白表达率低于无淋巴结转移组 (P <0 0 5 )。结论 :cyclinE蛋白高表达与 p2 1WAF1/CIP1蛋白失表达可能参与胃癌的发生发展过程 ,检测cyclinE和p2 1WAF1/CIP1蛋白作为反映胃癌病理学特点的参考指标可能有一定意义     

p21WAF1/Cip1 is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma

p21^WAF1/Cip1 is an inhibitor of cdk/cyclin complexes, and thus regulates the cell cycle. p21 is also related to cell differentiation and is regulated by wild-type p53, although p53-independent regulatory pathways have been proposed. In order to analyse p21 expression as well as its relationship with p53 in human breast cancer, an immunohistochemical analysis was undertaken of 77 breast carcinomas, 16 of them with an in situ component; 30 adjacent normal tissue samples; and five non-neoplastic specimens. Forty-four infiltrating carcinomas (57 per cent) were p21-positive. Expression of p21 was also observed in pre-invasive lesions, whereas normal ducts were negative or focally and weakly positive. p21 expression was associated with high histological grade (II+III) (P-0·017) and poor tubule formation (P-0·002), and was significantly less frequent in lobular carcinomas (P-0·0001). p21 positivity also correlated with increased proliferation, but this seemed to be dependent on the histological grade. Twenty carcinomas (26 per cent) showed p53 overexpression, but this was not associated with p21 negativity, suggesting the existence of p53-independent mechanisms for p21 regulation in vivo. Cyclin D1^CCND1 expression was analysed in the same series and an association between p21 and cyclin D1 expression was found, since 23 of 26 cyclin D1-positive carcinomas were p21-positive (P<0·001 …). In conclusion, p21 is frequently overexpressed in breast carcinomas and this occurs in the early stages of neoplastic progression. This overexpression seems to be independent of p53 status and might be involved in cyclin D1 modulation. © 1998 John Wiley & Sons, Ltd.     

Cisplatin causes cell death via TAB1 regulation of p53/MDM2/MDMX circuitry

The interdependence of p53 and MDM2 is critical for proper cell survival and cell death and, when altered, can lead to tumorigenesis. Mitogen-activated protein kinase (MAPK) signaling pathways function in a wide variety of cellular processes, including cell growth, migration, differentiation, and death. Here we discovered that transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1), an activator of TAK1 and of p38α, associates with and inhibits the E3 ligase activity of MDM2 toward p53 and its homolog, MDMX. Depletion of TAB1 inhibits MDM2 siRNA-mediated p53 accumulation and p21 induction, partially rescuing cell cycle arrest induced by MDM2 ablation. Interestingly, of several agents commonly used as DNA-damaging therapeutics, only cell death caused by cisplatin is mitigated by knockdown of TAB1. Two mechanisms are required for TAB1 to regulate apoptosis in cisplatin-treated cells. First, p38α is activated by TAB1 to phosphorylate p53 N-terminal sites, leading to selective induction of p53 targets such as NOXA. Second, MDMX is stabilized in a TAB1-dependent manner and is required for cell death after cisplatin treatment. Interestingly TAB1 levels are relatively low in cisplatin-resistant clones of ovarian cells and in ovarian patient''s tumors compared with normal ovarian tissue. Together, our results indicate that TAB1 is a potential tumor suppressor that serves as a functional link between p53–MDM2 circuitry and a key MAPK signaling pathway.     

Combined examination of p27(Kip1), p21(Waf1/Cip1) and p53 expression allows precise estimation of prognosis in patients with gastric carcinoma

AIMS: In order to estimate the prognostic values of p27(Kip1), p21(Waf1/Cip1), and p53, alone and in combination, we investigated immunohistochemically the expression of p27(Kip1), p21(Waf1/Cip1), and p53 proteins in gastric carcinomas. METHODS AND RESULTS: The expression of p27(Kip1), p21(Waf1/Cip1), and p53 was immunohistochemically examined in 140 gastric carcinomas. Positive expression of p27(Kip1) and p21(Waf1/Cip1) correlated significantly with a favourable prognosis (P < 0.05), whereas, positive expression of p53 tended to correlate with poor prognosis. Multivariate survival analysis revealed that TNM stage of tumour (P < 0.001), lymph node state (P=0.005), and p27(Kip1) expression (P=0.006) were independent prognostic factors. A striking stratification of mortality rate was found when patients were divided into four groups according to the expression of p21(Waf1/Cip1) and p27(Kip1). The mortality rate was higher in patients with both p21(Waf1/Cip1)- and p27(Kip1)-negative gastric carcinoma than in patients with one or both positive carcinomas (P < 0.01). In addition, if the four p21(Waf1/Cip1)/p27(Kip1) groups were compared based on p53 status, p53+ cases tended to have a higher mortality rate than p53- cases. CONCLUSION: Our results suggest that low expression of both p27(Kip1) and p21(Waf1/Cip1), could be useful as markers of poorer prognosis, and the combined examination of p27(Kip1), p21(Waf1/Cip1) and p53 expression allows reliable estimation of prognosis for patients with gastric carcinoma.     

Inactivation of p21WAF1/cip1 enhances intestinal tumor formation in Muc2-/- mice

In the Apc1638(+/-) mouse model of intestinal tumorigenesis, targeted inactivation of the cyclin-dependent kinase inhibitor p21(WAF1/cip1) is highly effective in enhancing Apc-initiated tumor formation in the intestine. Because p21(WAF1/cip1) plays a critical role in regulating intestinal cell proliferation, maturation, and tumorigenesis, we examined whether its inactivation would enhance tumor formation in a different mouse model of colon cancer. Therefore, we mated p21(-/-) mice with mice carrying a genetic deficiency of the Muc2 gene, which encodes the major gastrointestinal mucin. Muc2(-/-) mice develop tumors in the small and large intestine and the rectum, but in contrast to tumors in Apc1638(+/-) mice, this does not involve increased expression or nuclear localization of beta-catenin. We found that inactivation of p21(WAF1/cip1) significantly increased the frequency and size of intestinal tumors in Muc2 knockout mice and also led to development of more invasive adenocarcinomas. This enhanced tumorigenesis significantly decreased mouse life span. Further, inactivation of p21(WAF1/cip1) increased cell proliferation, decreased apoptosis, and decreased intestinal trefoil factor expression in the mucosa of both the small and large intestine. Surprisingly, reduced expression of p27(kip1) was also observed in the Muc2(-/-), p21(+/-), and p21(-/-) mice. In contrast, the expression of c-myc was significantly elevated. Thus, p21 modulates the formation of tumors whose initiation does (Apc) or does not (Muc2) involve altered beta-catenin-Tcf4 signaling, but which may converge on common elements downstream of this signaling pathway.     

LncRNA NCK1-AS1 promotes non-small cell lung cancer progression via regulating miR-512-5p/p21 axis

ObjectiveThis study aimed at probing into the effect of lncRNA NCK1-AS1 on proliferation, migration and invasion of non-small cell lung cancer (NSCLC) cells and its regulatory function on miR-512-5p/p21 molecular axis.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expressions of NCK1-AS1 and miR-512-5p in NSCLC tissues and cell lines. The alterations of cell proliferation, migration, invasion and cell cycle were examined by cell counting kit-8 (CCK-8) assay, BrdU experiment, Transwell experiment and flow cytometry, respectively. The dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the binding relationships between miR-512-5p and NCK1-AS1, and miR-512-5p the 3'UTR of p21 mRNA. Western blot was used to determine the effects of NCK1-AS1 and miR-512-5p on p21 protein expression.ResultsNCK1-AS1 expression was up-regulated in NSCLC tissues and cells, and its high expression was correlated with shorter overall survival time and faster progression of patients. Overexpression of NCK1-AS1 promoted NSCLC cell proliferation, migration and invasion, and accelerated the cell cycle, whereas NCK1-AS1 siRNA inhibited these malignant biological behaviors, and arrested cell cycle. NCK1-AS1 could bind to miR-512-5p, p21 was verified as a target gene of miR-512-5p, and NCK1-AS1 could up-regulate the expression of p21 in NSCLC cells via repressing miR-512-5p expression.ConclusionNCK1-AS1 promotes NSCLC progression by regulating miR-512-5p/p21 molecular axis.     

Expression of p21WAF1/CIP1 in colorectal adenomas and adenocarcinomas and its correlation with p53 protein expression

The expression of p53-Inducible cylln-dependent kinase Inhibitor, p21^WAF1/CIP1 in non-neopiastic mucosa, adenoma and adenocarclnoma of the colorectum was examined by immunohistochemistry and western bootting and Its relation with the expression of p53 protein was analyzed. Non-neoplastic epithelial cells at the surface area showing no proitferative activity expressed p21^WAF1/CIP1.The expression of p21^WAF1/CIP1 was lmmunohistochemlcally detected in 55% (206/377 of the adenomas and 66% (190/289) of the adenocarcinomas, respectively. The lncldence of strongly positive cases was significantly higher In the adenocarcinomas (27%) than In the adenomas (18%) ( P< .05). The incidence of cases wtth strong p21^WAF1/CIP1 expression was higher In stages 0,1 and 2 carcinomas than in stages 3 and 4 carcinomas ( P <0.05). A decrease in the incidence of cases with strong expression was detected in carclnomas Invading deeper than muscularis propria. The influence of strongly positive cases was signiflcantly lower in carcinomas with lymph node metastasis than those without metastasls ( P <0.05). The expression of p21 as well as p53 detected by western blotting was compatlble with the results of lmmunohistochemlstry in most cases examined. However, there was no significant correlatlon between the expression of p21^WAF1/CIP1 and the abnormal accumulation of p53. These findlngs overall suggest that: (i) the physiological expression of p21^WAF1/CIP1 may be associated with cellular senescence of colorectal mucosa; (ii) reduced expression of p21^WAF1/CIP1 participate in the progression of colorectal carcinoma; and (iii) p53-Independent paulway may be considerably Involved In the inductions of p21^WAF1/CIP1.     

p21WAF1/CIP1 expression in colorectal carcinoma correlates with advanced disease stage and p53 mutations

Defects in the mechanisms controlling the cell cycle are crucial in cell transformation and/or tumour progression. p21^WAF1/CIP1 is an inhibitor of cyclin-dependent kinases, induced by p53-dependent and p53-independent pathways, which can block progression through the cell cycle. p21^WAF1/CIP1 expression has been investigated immunohistochemically in a series of 191 patients with colorectal cancer of known p53 status. The purpose of the study was two-fold: to assess the relationship between p21^WAF1/CIP1 immunoreactivity and p53 alterations, and to evaluate the prognostic significance of p21^WAF1/CIP1 expression. In 96 carcinomas (51 per cent), p21^WAF1/CIP1 was expressed in over 10 per cent of tumour cells, whereas in 26, p21^WAF1/CIP1 was detected in under 10 per cent of neoplastic cells; 69 tumours lacked p21^WAF1/CIP1 expression. Immunoreactivity was more frequent in tumours of the right colon (p < 0·003) and was inversely correlated with tumour stage (p < 0·03), p53 gene mutations (p < 0·0007), p53 protein accumulation (p < 0·019), and Bcl-2 expression (p < 0·0005). In univariate analysis, down-regulation of p21^WAF1/CIP1 expression was associated with poor overall (p = 0·0022) and disease-free survival (p = 0·0009). Multivariate analysis, however, did not confirm any independent prognostic significance of p21^WAF1/CIP1 expression. The results indicate that p21^WAF1/CIP1 is associated with abnormal accumulation of p53 protein and the occurrence of p53 gene mutations in colorectal cancer and that lack of p21^WAF1/CIP1 expression is correlated with reduced patient survival in univariate analysis. These data underline the crucial pathogenetic role of the p53–p21^WAF1/CIP1 pathway in carcinomas of the large bowel. Copyright © 1999 John Wiley & Sons, Ltd.     

Radiation-Induced p53 and p21WAF-1/CIP1 Expression in the Murine Intestinal Epithelium : Apoptosis and Cell Cycle Arrest

p53-dependent expression of p21^WAF-1/CIP1 has been studied in murine intestinal epithelium after exposure to ionizing radiation. In un-irradiated small intestine, neither p53 nor p21^WAF-1/CIP1 could be detected by immunohistochemistry. After irradiation (8 Gy), there was a time- and dose-dependent increase in the expression of both proteins. In the small bowel, the positional expression of p53 and p21^WAF-1/CIP1 was similar but not coincident. Both proteins could be observed throughout the crypts with greatest frequency of expression over the first 15 cell positions, which includes the stem cell population (approximately positions 3 to 5) and the proliferating, transit cell population (approximately positions 5 to 15). p53-positive cells were primarily distributed toward the base of the crypt relative to p21^WAF-1/CIP1. Subdivision of the p53-positive cell population revealed that the cells with strongest p53 immunoreactivity were positioned farther toward the base of the crypt, and their distribution was approximately coincident with the frequency distribution of apoptotic cells. Cells that were either weakly or moderately immunoreactive for p53 were located toward the middle of the crypt and were approximately coincident with the distribution of p21^WAF-1/CIP1. The numbers of both p53- and p21^WAF-1/CIP1-positive cells declined steadily with time, and by 6 days after irradiation there were very few immunoreactive cells to observe. Radiation-induced increase in p53 and p21^WAF-1/CIP1 expression was not detected in mice homozygously null for p53. Expression of p21^WAF-1/CIP1 and incorporation of tritiated thymidine were found to be mutually exclusive. In the large bowel, p21^WAF-1/CIP1 and p53 expression were observed along the entire length of the colonic crypts after irradiation (8 Gy), and, unlike in the small intestine, this expression was not only maintained but increased over 72 hours. p21^WAF-1/CIP1 immunoreactivity was detected in large intestine epithelium up to 6 days after irradiation. The differential expression of p21^WAF-1/CIP1, observed between the large and small bowel and within the small intestinal crypts, is discussed.