Tomomi M. Yamamoto

Determinants for Activation of the Atypical AGC Kinase Greatwall during M Phase Entry

ABSTRACT The atypical AGC kinase Greatwall (Gwl) mediates a pathway that prevents the precocious removal of phosphorylations added to target proteins by M phase-promoting factor (MPF); Gwl is thus essential for M phase entry and maintenance. Gwl itself is activated by M phase-specific phosphorylations that are investigated here. Many phosphorylations are nonessential, being located within a long nonconserved region, any part of which can be deleted without effect. Using mass spectrometry and mutagenesis, we have identified 3 phosphorylation sites (phosphosites) critical to Gwl activation (pT193, pT206, and pS883 in Xenopus laevis) located in evolutionarily conserved domains that differentiate Gwl from related kinases. We propose a model in which the initiating event for Gwl activation is phosphorylation by MPF of the proline-directed sites T193 and T206 in the presumptive activation loop. After this priming step, Gwl can intramolecularly phosphorylate its C-terminal tail at pS883; this site probably plays a role similar to that of the tail/Z motif of other AGC kinases. These events largely (but not completely) explain the full activation of Gwl at M phase.

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer.

AbstractmiRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA–target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

Regulation of Greatwall Kinase during Xenopus Oocyte Maturation

Greatwall kinase is required for M phase maintenance by inhibiting PP2A. Gwl associates with PP2A in G2 oocytes, but the complex dissociates during M phase (meiosis I). Mutating Lys71 to Met (K71M) generates gain-of-function Gwl kinase activity toward endosulfinethat is sufficient to induce oocyte maturation in the absence of progesterone.

Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen

Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor–positive (ER+) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer–associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Experimental Design: Tissues from patients with ER+ breast cancer were analyzed for the presence of CD146-positive (CD146pos) and CD146-negative (CD146neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells cocultured with CD146pos or CD146neg fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER+ breast cancer. Results: We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions: Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer and should be considered a target for drug development. Clin Cancer Res; 23(7); 1710–21. ©2016 AACR.

Regulation of the Aurora B chromosome passenger protein complex during oocyte maturation in Xenopus laevis.

ABSTRACT The dynamics of the Aurora B protein kinase during Xenopus oocyte meiotic maturation were examined. Resting G2 oocytes express inactive Aurora B that is not associated with other subunits of the chromosome passenger complex (CPC). Activity increases near the time of germinal vesicle breakdown in progesterone-treated oocytes, and this increase is correlated with the synthesis of inner centromere protein (INCENP) and survivin, components of the CPC. Ablation of INCENP synthesis led to the failure of progesterone treatment to activate Aurora B, but biochemical progression through the meiosis I-to-II transition and arrest at metaphase II were not affected. At fertilization, Aurora B was deactivated in concert with the degradation of INCENP, and the levels of Aurora B kinase activity and INCENP oscillated in subsequent embryonic cell cycles. Prevention of the decrease in Aurora B activity at fertilization by expression of ectopic wild-type INCENP, but not kinase-dead Aurora B INCENP, blocked calcium-induced exit from metaphase arrest in egg extracts.

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry.

Alternative Polyadenylation of PRELID1 Regulates Mitochondrial ROS Signaling and Cancer Outcomes

Disruption of posttranscriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the PRELID1 mRNA that enhances its steady-state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it has been demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell type–specific manner. Modulation of PRELID1 expression, including its posttranscriptional control, appears to be a common stress response across different cancer types. These data reveal that PRELID1 mRNA processing is an important regulator of cell type–specific responses to stress used by multiple cancers and is associated with patient outcomes. Implications: This study suggests that the regulation of PRELID1 expression, by APA and other mechanisms, plays a role in mitochondrial ROS signaling and represents a novel prognostic factor and therapeutic target in cancer. Mol Cancer Res; 15(12); 1741–51. ©2017 AACR.

Abstract P4-04-06: CD146 positive and negative stroma direct breast tumor estrogen receptor levels, therapeutic response and metastatic potential

Background: Cellular heterogeneity within all breast cancer subtypes remains a major cause of treatment failure and development of metastatic disease. Currently, both preclinical studies and drug development efforts focus almost exclusively on the epithelial component of breast cancers. Despite strong preclinical data novel therapies often fail in clinical testing. We propose that this failure is, in part, due to taking tumor cells out of their context and using pre-clinical models that fail to capture the complexity of human disease. We hypothesize that tumors hijack normal components of the tissue microenvironment and use it to their advantage. Here we demonstrate that similar to the normal hematopoetic niche, two major subtypes of breast cancer stroma can be defined by CD146 expression. We further show that the ratio of the stromal subtypes alters the response to therapy and increases the metastatic potential of breast cancer cells (BCC). Results: Tumor associated stroma, from all breast cancer subtypes, contains a mixture of CD146+ and CD146- fibroblasts. We isolated and derived pure human CD146+ and CD146- clonal lines. Both subtypes expressed markers of activated fibroblasts and clustered by gene expression profiling with normal stromal cell lines HS27A (CD146+) or HS5 (CD146-) according to CD146 expression. Although both stromal subtypes were derived from tumor associated tissue, CD146+ breast cancer stroma clustered with normal breast associated stroma and correlated with good clinical outcome (Finak et. al. Nature Med 2008). CD146- stroma clustered with breast cancer associated stroma and predicted worse outcome. Using cell line and patient-derived xenograft models of estrogen receptor (ER) positive breast cancer, we demonstrated that CD146+ compared to CD146- stroma supported significantly higher ER expression in BCCs. BCC co-cultured with CD146+ stroma responded more robustly to estrogen treatment and anti-endocrine therapy with tamoxifen. Next we used expression profiling data to predict stromal influence on treatment response. CD146+ stroma expressed 3-fold more TGFβ than CD146- stroma. Inhibiting TGFβ decreased proliferation 2-fold in BCCs grown in media conditioned by CD146+ stroma, but not by CD146- stroma. Conversely, HBEGF expression was 3-fold higher in CD146- stroma compared to CD146+ stroma. Inhibiting EGFR decreased proliferation 1.5-fold in BCCs grown in conditioned by CD146- stroma, but not by CD146+ stroma. In addition, intracardiac injection of stroma resulted in distant metastases in our primary orthotopic PDX model of breast cancer. Lastly we confirmed our preclinical observation using a small set of clinical samples. Patients with high CD146+ to CD146- stromal ratio had better clinical outcomes than patients with high CD146- to CD146+ stromal ratio. Conclusion: We conclude that stromal subtypes defined by CD146 expression direct the heterogeneity of ER expression, response to therapy and the metastatic potential of breast cancer cells. Citation Format: Heather M Brechbuhl, Jessica J Finlay-Schultz, Tomomi M Yamamoto, Austin E Gillen, Anthony Elias, Carol A Sartorius, Peter Kabos. CD146 positive and negative stroma direct breast tumor estrogen receptor levels, therapeutic response and metastatic potential [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-06.