Functional And Mechanistic Studies On Ras Related Aurora Kinase And Stanniocalcin 2 In Ovarian Tumorigenesis

Section Ⅰ. Ras related Aurora kinases controls ovarian and pancreatic tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidyBackgroundAurora kinases, a family of serine/threonine protein kinase that involved in cell mitosis, act downstream of Ras signalling pathway and are known as oncoproteins. Aurora kinases are highly expressed in various tumors. The activation of Aurora kinase A/B (AurA/B) or the inactivation of Breast cancer susceptibility gene 1/2 (BRCA1/2) often co-exist in ovarian and pancreatic cancer. The imbalance between AurA/B and BRCA1/2 may influence cancer cell growth and tumorigenesis, however, the interactive regulation between these molecules and the mechanisms manipulating tumorigenesis are still elusive.PurposeTo investigate the effects of the interplays between AurA/B and BRCA1/2 on tumorigenesis, which may help to optimize the diagnosis and treatments for cancers with imbalanced AurA/B and BRCAl/2.MethodSolo or consecutively silence the expression of AurA/B or/and BRCA1/2 with specific shRNAs in ovarian cancer cell OVCA433 and pancreatic cancer cell Capan-1, and detect the protein and mRNA levels of AurA/B or/and BRCA1/2 in resulting cells. Further investigate the cells behavior on cell proliferation and colony forming ability, cell cycle distribution and related proteins, cytokinesis, cell multinuclearity, chromosome tetraploidy and tumorigenicity in nude mice.ResultsIn ovarian cancer cell line OVCA433, depletion of AurA enhanced the expression of BRCAl/2, but not AurB, whereas interruption of AurB expression promoted the expressions of AurA and BRCA1/2. And in BRCA2 deficient pancreatic cancer cell Capan-1, the protein expression between AurA and AurB was complementary, and that the expression of BRCA1 was up-regulated after silencing of AurA or AurB, but knockdown of BRCA1 downregulated the expression of AurA. In both pancreatic and ovarian cancer cell lines, concurrent silencing of AurA and AurB dramatically elevated the expression of BRCA1/2, whereas double knockdown of BRCA1 and BRCA2 highly stimulated the expression of AurA, but not AurB. Solo or co-occurrent expression of different shRNAs target specifit genes did not the expression of other genes. Treating cells with MG132 eliminated the interplays between the AurA/B or/and BRCA1/2 proteins.Disruption of AurA and/or AurB inhibitied of cell proliferation, reduced the number of clones, retarded cell cycle progression, while inhibition of BRCA1 and/or BRCA2 promoted cell proliferation, increased the number of colony formation and promoted cell cycle progression. Co-occurrent inhibition of AurA/B and BRCA1/2 counteract the effects of AurA/B or BRCA1/2 on cell proliferation, colony formation and cell cycle distribution.Silencing AurA/B and/or BRCA1/2 altered the expression of a series of cell cycles related proteins, among which the changes of p53 and cyclinA were most consistent among different cells. Disruption of AurA and/or AurB increased p53 but decreased cyclin A, while inhibition of BRCA1 and/or BRCA2 reduced p53 but elevated cyclin A, while co-silencing of AurA/B and BRCA1/2 offsetted the individual impacts of AurA/B or BRCA1/2 on p53 and cyclin A.Compared with the corresponding control cells, cells with low levels of AurA and/or AurB exhibited less multinuclearity and chromosome tetraploidy, while cells expression BRCA1 and/or BRCA2 shRNAs showed more multinuclearity and tetraploidy. Cells with low levels of all the four proteins had equivalent levels of chromosomal multinucleated cells and tetraploid chromosomes with corresponding control cells. Furthermore, in telophase, AurA/B and BRCA1/2 co-localized at the midbody in both cells, but the accumulation of BRCA1 at the midbody was not obvious. Inhibition of AurA and/or AurB increased the accumulation of BRCA2 at the midbody, but not BRCA1. And disruption of BRCA1 and or BRCA2 increased the accumulation of AurA/B at the midbody.Compared with the corresponding control cells, the tumor with lower levels of AurA and/or AurB grew slower, while tumors with lower expression BRCA1 and/or BRCA2 grew fester. Tumors with lower levels of all the four proteins grew fester than tumors with lower levels of AurA and AurB. but slower than tumors with BRCA1 and BRCA2.ConclusionThe negative interplays between Aur A/B and BRCA1/2 may inversely control cell proliferation, cell cycle progression, cytokinesis, cell multinuclearity, and tetraploidization to modulate tumorigenesis.Section Ⅱ. Stanniocalcin 2 and Ovarian Tumorigenesis BackgroundThe secreted protein Stanniocalcin (STC) are involved in regulating calcium and phosphorus homeostasis in fish. Human STCs contains two members, STC1 and STC2, both of which are related with various human cancer. STC1 are elevated in RAS transformed ovarian cancer cells and promote tumorigenesis, but the role of STC2 in ovarian cancer needs to be illustrated.PurposeTo study the effect and mechanism of STC2 in the occurrence and progression of ovarian cancer, trying to provide valuable views for the diagnosis and treatments for ovarian cancer.MethodsOverexpressing HA-tagged STC2 in ovarian cancer cell line SKOV3, to detect whether the secreted protein STC2 could act on its source cells; Silencing or overe xpressing of STC2 with shRNA or cDNA in a series of ovarian cancer cell lines, to systematically investigate the effects of STC2 on cell proliferation, plate clone formatioin, cell cycle distribution, the sencitivity to cisplatin and paclitaxol, cell migration and invasion in ovarian cancer cells; Building animal models, to observe the effects of STC2 in vivo; Finally, building tissue chips of human ovarian carcinoma and normal ovarian, analyzing the relationship between STC2 expression and patient survival.ResultsHA-tagged STC2 could be secreted out to the extracellular medium, and then binded on the cell membrane. In Hey, Hey A8, OVCA420, OVC A429 cells, compared with corresponding control cells, silencing of STC2 inhibited cell proliferation and colony formation, promoted G1-S checkpoint arrest, enhanced cell sensitivity to cisplatin and paclitaxel, but promoted cell migration and invasion. On the contrary, in A2780, SKOV3, SKOV3 ipl cells, compared with corresponding control cells, overexpressing of STC2 promoted cell proliferation and colony formation, promoted G1-S transition, inhibited the apoptosis in response to cisplatin and paclitaxel, but inhibited cell migration and invasion. Compared with corresponding control cells, cells with lower levels of STC2 exhibited lower levels of cell cycle related proteins CDK2, CDK4 and CDK6, but higher levels of pro-apoptotic protein Bax, Bid and cytoC, higher levels of the anti-survival but pro-metastasis protein p38, pp38, and higher or unchanged levels of STAT3 and lower levels of pSTAT3, with unchanged levels of MEK, ERK1 and pERK1/2. The levels of proteins in cells with overexpressing STC2 altered on the opposite trends. Disruption of STC2 expression in Hey-STC2i cells inhibited the growth of subcutaneously grafted tumor in nude mouse compared with Hey-Scr cells, while overexpression of STC2 in SKOV3 ip1-STC2 cells promoted tumor growth compared with SKOV3 ip1-Vector cells; on the other hand, silencing of STC2 promoted peritoneal metastasis in nude mouse, but overexpression of STC2 inhibited peritoneal metastasis. STC2 was highly expressed in ovarian cancer tissue, but could not be detected in normal ovarian tissue. And patients with higher expression of STC2 had longer Progression Free survival and Overall Survival.ConclusionSTC2 may promote cell proliferation and drug resistance in ovarian cancer cells and promote tumor growth, but may suppress ovarian cancer cells migration, invasion and further tumor metastasis, and higher expression of STC2 in ovarian cancer may be related with better prognosis.