Summary Regulators of mitosis have been successfully targeted to enhance response to taxane chemotherapy. Here, we show that the Salt Inducible Kinase 2 (SIK2) localizes at the centrosome, plays a key role in the initiation of mitosis and regulates the localization of the centrosome linker protein, C-Nap1, through S2392 phosphorylation. Interference with the known SIK2 inhibitor PKA induced SIK2-dependent centrosome splitting in interphase while SIK2 depletion blocked centrosome separation in mitosis, sensitizing ovarian cancers to paclitaxel in culture and in xenografts. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher expression of SIK2 significantly correlated with poor survival in patients with high-grade serous ovarian cancers. These data identify SIK2 as a plausible target for therapy in ovarian cancers.

BACKGROUND Trastuzumab is part of the standard treatment for HER-2 positive breast cancer patients, but not all patients respond to trastuzumab. Altered expression levels for microRNAs in cancer cells have been correlated with prognosis and response to chemotherapy. We hypothesized that altered expression levels for miRNAs in plasma are associated with sensitivity to trastuzumab in patients with HER-2 positive breast cancer. METHODS We performed quantitative RT-PCR in plasma samples including breast cancer patients enrolled in a clinical trial of neoadjuvant trastuzumab-based chemotherapy. We analyzed expression levels for miR-210, -21, -29a, and -126 according to the type of response (pCR (n = 18) vs. residual disease (n = 11)). We also compared expression levels of miRNAs in trastuzumab-sensitive and –resistant breast cancer cells derived from BT474 cells and in an independent set of preoperative (n=39) and postoperative plasma (n=30) from 43 breast cancer patients not given any treatment. RESULTS At baseline before neoadjuvant chemotherapy combined with trastuzumab, circulating miR-210 levels were significantly higher in patients who had residual disease than in those who had pathologic CR (P = 0.0359). Mean expression ratio for miR-210 was significantly higher in trastuzumab-resistant BT474 cells and miR-210 expression was significantly higher before surgery than after surgery (P = 0.0297) and in patients whose cancer metastasized to the lymph nodes (P = 0.0030). CONCLUSIONS Circulating miR-210 levels were associated with trastuzumab sensitivity, tumor presence, and lymph node metastases. This suggests that plasma miR-210 may be used to predict and perhaps monitor response to therapies containing trastuzumab.

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin s ) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro-and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.

Cyclin-dependent kinase (CDK) inhibitor p27

The promyelocytic leukemia gene (PML) involved in the t(15;17) (q22;q12) translocation in acute promyelocytic leukemia is a growth suppressor. To elucidate the functional domains of PML, several mutants lacking the nuclear localization signal (PMLnls-), the dimerization domain (PMLdim-), the proline-rich domain at the N-terminal (PMLpro-), the proline-rich RING finger motif (PMLpr-), the proline-rich RING finger B-box-1 (PML-prb-), the serine-proline-rich domain at the C-terminal (PMLsp-), and the double mutant (PMLprb-nls-) have been constructed. Immunofluorescence staining of transiently transfected NIH3T3 cells demonstrated that the RING finger motif, dimerization domain, and nuclear localization signal are all required for the formation of PML oncogenic domains (PODs). Immunofluorescence staining of transiently transfected GM637D human fibroblasts indicated that expression of PMLprb-, PM-Lnls-, and PMLprb-nls- led to a significant reduction or, in some cases, complete elimination of PODs. PMLdim-, PMLnls-, PMLpr-, PMLprb-, and PMLprb-nls- mutants were found to lose their ability to suppress transformation of NIH3T3 cells by activated neu, while PMLpro- and PMLsp- mutants did not. These results suggest that the ability of PML to form a POD is essential for suppression of growth and transformation. Furthermore, since PMLprb-, PMLnls-, and PMLprb-nls- mutants could block the suppression effect of wild-type PML on transformation of NIH3T3 cells by the neu oncogene, these PML mutants are potential dominant negative inhibitors of PML. Our study also suggests that the RING finger motif may interact with other nuclear proteins.