Transient arrest in a quiescent state allows ovarian cancer cells to survive suboptimal growth conditions and is mediated by both Mirk/dyrk1b and p130/Rb2

Abstract

Some ovarian cancer cells in vivo are in a reversible quiescent state where they can contribute to cancer spread under favorable growth conditions. The serine/threonine kinase Mirk/dyrk1B was expressed in each of seven ovarian cancer cell lines and in 21 of 28 resected human ovarian cancers, and upregulated in 60% of the cancers. Some ovarian cancer cells were found in a G0 quiescent state, with the highest fraction in a line with an amplified Mirk gene. Suboptimal culture conditions increased the G0 fraction in SKOV3 and TOV21G, but not OVCAR4 cultures. Less than half as many OVCAR4 cells survived under suboptimal culture conditions as shown by total cell numbers, dye exclusion viability studies, and assay of cleaved apoptotic marker proteins. G0 arrest in TOV21G and SKOV3 cells led to increased levels of Mirk, the CDK inhibitor p27, p130/Rb2, and p130/Rb2 complexed with E2F4. The G0 arrest was transient, and cells exited G0 when fresh nutrients were supplied. Depletion of p130/Rb2 reduced the G0 fraction, increased cell sensitivity to serum-free culture and to cisplatin, and reduced Mirk levels. Mirk contributed to G0 arrest by destabilization of cyclin D1. In TOV21G cells, but not in normal diploid fibroblasts, Mirk depletion led to increased apoptosis and loss of viability. Because Mirk is expressed at low levels in most normal adult tissues, the elevated Mirk protein levels in ovarian cancers may present a novel therapeutic target, in particular for quiescent tumor cells which are difficult to eradicate by conventional therapies targeting dividing cells.Normal diploid cells such as lymphocytes and fibroblasts go into a reversible growth arrest called quiescence in response to anti-proliferative environmental signals. These normal cells maintain quiescence through upregulating the CDK inhibitor p27 in part through increasing the stability of the CDK inhibitor p27 through the kinase Mirk, 1-3 upregulate the transcriptional repressor HES1 to block senescence or differentiation, 4,5 and block apoptosis through several mechanisms. A subpopulation of tumor cells, possibly including stem cells, remains in a nondividing quiescent state that renders them relatively resistant to chemotherapy and radiation therapy which target dividing cells.6 By entering a quiescent state, tumor cells can resist the nutrient deficiencies, hypoxic and acidic conditions within the tumor mass. Mirk has also been reported by our group to maintain the viability of quiescent pancreatic cancer cells by reducing intracellular levels of reactive oxygen species (ROS). 7 Normal cells in quiescence activate pathways that protect them from metabolic stress, and a subpopulation of tumor cells is likely to utilize similar pathways to survive within the tumor microenvironment. The anti-apoptotic proteins BCL2 and BCL-XL have been shown to facilitate G0 quiescence in murine embryonic fibroblasts by decreasing RNA content and cell size, and by upregulating p27 protein through its stabilization following phosphorylation of p27Ser10 by Mirk. 3 Epit...

Mirk/dyrk1B kinase is upregulated following inhibition of mTOR

“…However, Mirk kinase expression and activity are highest when cells are out of cycle in a quiescent state (14,22), when cAMP levels are elevated. Given that the Mirk promoter has potential CREB transcription factor binding sites, the cyclic adenosine monophosphate (cAMP) response element binding protein CREB was investigated.…”

“…The results of the current study suggest that an additional mediator of cell survival is Mirk/dyrk1B, a kinase with reactive oxygen species (ROS)-suppressing functions in pancreatic, ovarian and colon cancers (11–13). Mirk/dyrk1B was expressed in 21 of 28 (75%) resected human ovarian cancers, primarily papillary serous cystadenocarcinomas, with upregulation in 60% of the cancers (14). In a larger clinical screen of 76 patient samples, Mirk protein was detected in 75% of the cancers and overexpressed in 41%, with lower incidence in the benign tumors and none in the non-neoplastic ovarian cysts (15).…”

“…Also, 86% of serum-starved HD6 colon carcinoma cells accumulated in G 0 compared with 14% when Mirk was depleted (19). Suboptimal growth conditions would normally signal entry of many cancer cells into G 0 if Mirk was active, and cells cycled out of G 0 when normal serum levels were restored, showing the entry into G 0 was reversible (11,13,14). However, if Mirk was depleted or inactivated, many serum-starved TOV21G or SKOV3 ovarian cancer cells or Panc1 or SU86.86 pancreatic cancer cells underwent apoptosis instead of remaining viable in G 0 .…”

“…Thus, Mirk/dyrk1B is a kinase active in quiescent ovarian, colon or pancreatic cancer cells, so presents an attractive drug target in these cells. Mirk levels vary up to 10-fold during the cell cycle (16,22), reaching their peak when cells become quiescent in response to energy limitation caused by nutrient or serum starvation (14,21), but the mechanisms that upregulate Mirk expression in quiescent cells are unknown. Signaling from mTOR (mTORC1) activates steps in translation and metabolism essential for cell growth.…”

Biological Consequences of Priming Phosphorylation in Cancer Development

“…In contrast, DYRK1B (also known MIRK), closely related to DYRK1A, have been characterized as a positive regulator of cancer cell survival [50][51][52][53][54][55][56][57][58][59][60][61]. It is still not known to the details of the biological functions for DYRK3 and DYRK4.…”