Oncogenic RAS Induces Accelerated Transition through G2/M and Promotes Defects in the G2 DNA Damage and Mitotic Spindle Checkpoints

Abstract

Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS V12 increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS V12 or HRAS V12 effector mutants were presynchronized at the G 1 /S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS V12 accelerated the G 2 /M phase by ϳ4 h and promoted bypass of the G 2 DNA damage and mitotic spindle checkpoints. Accelerated passage through G 2 /M and bypass of the G 2 DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G 2 DNA damage checkpoint, because cells arrested appropriately in G 2 despite conditional expression of HRAS V12,S35 or BRAF V600E . By contrast to the MAPK requirement for radiation-induced G 2 arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G 2 progression and regulation of the G 2 DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G 2 /M.Human tumors, including those of the thyroid (1-3), arise from a single transformed cell. Despite their clonal origin, cells from advanced carcinomas are often genetically heterogeneous. Tumor cell variability is thought to result from genomic instability. Clonal heterogeneity in turn tends to predict a poor outcome and resistance to therapy. This is also the case in thyroid cancer. In thyroid tumors, the nature of the oncogenic events involved in the initiation of the neoplastic clone may determine the likelihood of genomic instability occurring at a later stage. Among the various forms of thyroid neoplasia, follicular adenomas and carcinomas are commonly aneuploid, whereas abnormalities in chromosome number are comparatively less frequent in papillary thyroid carcinomas. Mutations of all three RAS genes are found in benign and malignant follicular neoplasms and in follicular variant papillary thyroid carcinomas and are believed to be one of the early steps in thyroid tumor formation (4 -9). By contrast, rearrangements of the tyrosine kinase receptor gene RET (rearranged by transfection; RET/ PTC) are only found in papillary thyroid carcinomas, which are commonly diploid and less aggressive. A possible explanation for this is that activating mutations of RAS, but not of RET, promote tumor progression in part by decreasing genomic stability. Consistent with this is the observation that ...

Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

“…Further, MAPK are known to participate in the maintenance of G2/M arrest and they are required for exit from growth arrest and transition through mitosis. 26 In our previous study, we had found that Ras can induce CARF, and we show here that CARF inhibition reciprocally decreases Ras, leading to downregulation of the Ras-mediated MAPK pathway, possibly as a measure to halt the cell cycle. Nonetheless, using two independent assays including ERK-overexpressing and HT1080 cells, we found that the Ras-MAPK pathway is not essential for CARFinhibition-induced apoptosis.…”

Upregulation of miR-21 by Ras in vivo and its role in tumor growth

“…Genes regulated by miR-21 in thyroid cells are enriched in categories involved in cell cycle and DNA damage response, especially by looking selectively at downregulated genes. It has been reported that oncogenic Ras is able to induce chromosome instability and to accelerate G2/M transition (Abulaiti et al, 2006;Knauf et al, 2006), thus we hypotesize that miR-21 could contribute to the ability of Ras-transformed cells to overcome the G2 DNA damage checkpoint successfully. We plan to perform additional experiments to verify this hypothesis.…”

Role of MEK/ERK pathway in the MAD2-mediated cisplatin sensitivity in testicular germ cell tumour cells

“…In addition, the localisation of active ERK to the mitotic kinetochore is also suggested to regulate proteins involved in chromosome segregation during metaphase to anaphase transition (Shapiro et al, 1998;Zecevic et al, 1998). Furthermore, ERK is also described to associate with kinetochores during early prophase, but this association is not apparent at later stages of mitosis (Knauf et al, 2005). The fact that MAD2 functions through its localisation to kinetochore suggests that there may be a direct interaction between the MEK/ERK pathway and MAD2 in regulating mitosis.…”