The Mechanism Of P53^N236S Cooperates With Oncogenic Ras In Promoting Tumorigenesis

Most normal human cells have limited level of telomerase activity and are lack of telomere maintains mechanism, which resulted in telomere loss and replicative senescence. There is a strong correlation between senescence and cacinogenesis. On the one hand, accelerated aging drives accumulation of DNA mutation, genome instability, telomere dysfunction etc., fueling tumor progression and metastasis. On the other hand, like apoptosis, senescence is considered as an important barrier that limits the proliferative potential of cells with DNA damages resulted from inner or outer stresses. Therefore, tumorigenesis is the result of lost the regulation of DNA damage monitoring mechanism such as cellular senescence, apoptosis. In addition, tumor cells need to reactivate the telomere maintenance mechanism in order to avoid telomere shortening during cell proliferation. About85-90%tumors could lengthen telomere by reactivating telomerase. Instead of reactivating telomerase,10-15%tumors maintain their lengths of telomeres by the mechanisms referred as alternative lengthening of telomere (ALT). Since the ALT tumor is telomerase-negative, the research on ALT tumor will facilitate the understanding of the interaction between aging and carcinogenesis. What's more, in a large number of tumor treatment strategies which aimed to inhibit telomerase activity, tumor cells may be forced to choose ALT as telomere maintenance mechanism to maintain their proliferation. Therefore, the studies on ALT may also be important for diagnosis and treatment of telomerase-positive cancer.In this study, we aimed to understand the loss and gain of function of a mutant p53protein (p53N239S in human, p53N236S in mouse, or p53S) in ALT tumor. As a tumor-suppressor gene, p53plays a very important role in inhibiting tumorigenesis by inducing cell cycle arrest and apoptosis in response to various types of stress. In about80%ALT tumors, p53were found mutated. IARC database showed that p53S was found widely in32human tumor cases.Our previous studies have established7strains of immortalized cells derived from Werner syndrome(WS) mouse model G5mTerc-/-Wrn-/-MEFs(mouse embryo fibroblast). In a comparative study of three strains of ALT tumorigenic immortalized cell lines(3B-1,3B-2,9B-1) and four strains of Non-ALT tumorigenic cell lines(7A-1,7A-2,7A-3,6B-1), a p53mutant p53N236S (p53N239S in human, p53S) was found highly expressed in these three independent ALT tumorigenic immortalized cell lines. Computational structure analysis suggested that p53N236S could cause the reformation of hydrogen bonds in the local area, and lead to collapse of DNA binding conformation. The Western blot analysis of protein expression profile in3B-1,3B-2and9B-1cell lines indicated high expression level of p53N236S and phosphorylated p53correlated with the low expression level of p21Cipl/Wafl, suggesting the mutation of p53protein and dysfunction of the p53pathway occurring in ALT tumorigenic cells. Furthermore, real-time PCR confirmed that the p53N236S has lost the function of regulating the transcription of p21CiP1/Wafl, cyclin G, PUMA, Bax in response to1OGy irradiation. These data suggested that p53S lost transcriptional regulatory function in both cell cycle arrest and apoptotic pathways, thus lost the function of inhibiting tumorigenesis. To our surprise, introducing of p53S into p53-/-MEFs (p53-/-+S) did not generate tumors by subcutaneously injecting the cells into SCID mouse, suggesting that p53S per se is not enough to be tumorigenic. However, cooperating with H-RasG12V(p53-/-+S+Ras), p53N236S could dramatically promote tumorigenesis in p53null MEFs when subcutaneously injecting the cells into SCID mouse, strongly suggesting a gain of function of p53S cooperating with H-RasG12v in tumorigenesis. Supporting this, we also found that co-expression of p53S and H-RasG12V could partially eliminate the activation of stress response caused by either p53S or H-RasG12V, which might facilitate the cell growth and carcinogenesis. Flow cytometry analysis showed that Iess p53-/-+S+Ras cells were located in G1/G0phase and more in S phase than p53V-/-+vector+Ras cells. This result further suggested a cooperation between p53S and H-RasG12V in promoting cell growth. Chromosome analysis revealed dramatically increase of double minutes and aneuploidy (2N>40) in p53-/-+S+Ras,P53-/-+vector+Ras tumor cells and P53-/-+S MEFs in contrast with p53-/-MEFs and wild type MEFs. This result suggested p53S cooperate with H-RasG12V in promoting the genome instability. Together these results indicated the gain of function of p53S in cooperating with H-RasG12V and supported the oncogenic effect of p53mutants in ALT tumorigenesis.Supported by NSFC grants, we successfully established the p53N236S gene knock-in mouse model. The homozygous (p53S/S) and heterozygous (p53S/+) p53S MEF cell lines were established based on this model. When transiently transfected p53S/+and p53+/+MEFs with H-RasG12V, a lot of senescent cells emerged in both p53S/+and p53+/+cells, showing slow proliferation and flat morphology. The SA-β-Gal staining showed approximately68%cells undergo senescence in the p53S/+transiently transfected with H-RasG12V. However, when P53S/+cells expressing H-RasG12V were cultured to10passages, senescent cells decreased rapidly. And the late generation of p53S/++H-RasG12V cells could generate tumors by subcutaneously injecting into SCID mouse, suggesting the p53S heterozygote have lost the wild-type allele when exposed to high level of H-RasG12V. Interestingly, when we genotyped the late generation of p53S/++H-RasG12V cells, the amount of the wild type DNA band decreased obviously. This result suggested that the p53wild type allele may be deleted by the loss of heterozygosity (LOH) mechanism. To further dissect the molecular basis of the p53LOH under the condition of high level H-RasG12V, we analyzed the expression of proteins in three MEF cell lines:p53S/+, p53+/+and p53S/S transfected with H-RasG12V on a time series. In both p53+/+and p53S/+cells, the p21protein level was up-regulated24hr after H-RasG12V transfection. Interestingly, in p53S/+cells stably transfected with H-RasG12V (1month after transfection), p21protein level was a bit down-regulated comparing with transient transfection. At the same time, p53, phosphorylated p53, Hsp70and p19ARF level are elevated rapidly. These results suggest that, in P53S/+cell, transient expression of H-RasG12V oncogenic signal could not induce tumorigenesis. However, when the proliferative signal persisted for a certain period of time, p53S/+cells will escape from senescence and activate tumor progression. At the last, we found p53S/+cells with exogenous expressed H-RasG12V are more resistant to doxorubicin, a DNA damage reagent. These results suggested the overexpression of H-RasG12v oncogenic signal causes a LOH in heterozygous (p53S/+) p53S MEF cells, ultimately result in tumorigenesis.As a conclusion, the p53N236S was found in three independent ALT tumorigenic cell lines derived from senesced Werner Syndrome MEFs. During the study of the characteristic of p53S, we have observed that p53S lost transcriptional regulatory function in both cell cycle arrest and apoptotic pathways, and gained new function in promoting tumorigenesis in vivo when cooperates with oncogenic Ras. In the cells with endogenous p53S expression, the overexpression of H-RasG12v oncogenic signal cause a LOH of wild-type allele in heterozygous p53S MEF cell (p53S/+), result in escaping from senescence and activate tumor progression._The research on structure and GOF of p53N236S may help to understand the initiation and progression of ALT tumor.