Hippo pathway target, YAP has emerged as an important player in solid tumor progression. Here, we identify RUNX1 and RUNX3 as novel negative regulators of oncogenic function of YAP in the context of breast cancer. RUNX proteins are one of the first transcription factors identified to interact with YAP. RUNX1 or RUNX3 expression abrogates YAP-mediated pro-tumorigenic properties of mammary epithelial cell lines in an interaction dependent manner. RUNX1 and RUNX3 inhibit YAP-mediated migration and stem-ness properties of mammary epithelial cell lines by co-regulating YAP-mediated gene expression. Analysis of whole genome expression profiles of breast cancer samples revealed significant co-relation between YAP–RUNX1/RUNX3 expression levels and survival outcomes of breast cancer patients. High RUNX1/RUNX3 expression proved protective towards YAP-dependent patient survival outcomes. High YAP in breast cancer patients’ expression profiles co-related with EMT and stem-ness gene signature enrichment. High RUNX1/RUNX3 expression along with high YAP reflected lower enrichment of EMT and stem-ness signatures. This antagonistic activity of RUNX1 and RUNX3 towards oncogenic function of YAP identified in mammary epithelial cells as well as in breast cancer expression profiles gives a novel mechanistic insight into oncogene–tumor suppressor interplay in the context of breast cancer progression. The novel interplay between YAP, RUNX1 and RUNX3 and its significance in breast cancer progression can serve as a prognostic tool to predict cancer recurrence.

Persistent activation of signal transducer and activator of transcription 3 (STAT3) is associated with the progression of a range of tumors. In this report, we present the anticancer activity of 2-(1-(4-(2-cyanophenyl)1-benzyl‑1H-indol-3-yl)-5-(4-methoxy-phenyl)-1-oxa-3-azaspiro(5,5)undecane (CIMO) against breast cancer cells. We observed that CIMO suppresses the proliferation of both estrogen receptor-negative (ER-) (BT-549, MDA-MB‑231) and estrogen receptor-positive (ER+) (MCF-7, and BT-474) breast cancer (BC) cells with IC50 of 3.05, 3.41, 4.12 and 4.19 µM, respectively, and without significantly affecting the viability of normal cells. CIMO was observed to mediate its anti-proliferative effect in ER- BC cells by inhibiting the phosphorylation of JAK2 and STAT3 proteins. Quantitative PCR analysis demonstrated that CIMO decreases the relative mRNA expression of genes that are involved in cell cycle progression (CCND1) and cell survival (BCL2, BCL-xL, BAD, CASP 3/7/9, and TP53). In addition, CIMO was observed to arrest BC cells at G0/G1 phase and of the cell cycle. Furthermore, CIMO suppressed BC cell migration and invasion with concordant regulation of genes involved in epithelial to mesechymal transition (CDH1, CDH2, OCLN and VIM). Thus, we report the utility of a synthetic azaspirane which targets the JAK-STAT pathway in ER- BC.

Background Germinomas (IG) account for up to 50% of all intracranial germ cell tumours. These tumours are reputed to be more prevalent in Oriental populations in comparison to Western cohorts. Biological characteristics of IG in other ethnic groups are unknown. Singapore is a multi-ethnic country with diverse cultures. Owing to inter-racial heterogeneity, the authors hypothesize there are molecular differences between paediatric IG patients in our local population. The aims of this study are exploratory: firstly, to identify molecular characteristics in this tumour type and circulating CSF unique to different racial cohorts; and next, to corroborate our findings with published literature. Methods This is a single-institution, retrospective study of prospectively collected data. Inclusion criteria encompass all paediatric patients with histologically confirmed IG. Excess CSF and brain tumour tissues are collected for molecular analysis. Tumour tissues are subjected to a next generation sequencing (NGS) targeted panel for KIT and PDGRA. All CSF samples are profiled via a high-throughput miRNA multiplexed workflow. Results are then corroborated with existing literature and public databases. Results In our cohort of 14 patients, there are KIT exon variants in the tumour tissues and CSF miRNAs corroborative with published studies. Separately, there are also KIT exon variants and miRNAs not previously highlighted in IG. A subgroup analysis demonstrates differential CSF miRNAs between Chinese and Malay IG patients. Conclusion This is the first in-depth molecular study of a mixed ethnic population of paediatric IGs from a Southeast Asian cohort. Validation studies are required to assess the relevance of novel findings in our study.

Triple-negative breast cancer (TNBC) is considered one of the aggressive breast cancer subtypes with higher risk of recurrence and mortality compared to other subtypes. There is a need to identify molecular markers and drivers that could distinguish metastatic subset of TNBC. Based on our previous study (Kulkarni et al., Oncotarget 2018), we investigated for association of elevated YAP expression specifically in TNBC tissue samples and cell lines. Six TNBC cell lines with different degrees of YAP expression were selected for the study. Cell lines were grouped based on high, intermediate, or low nuclear YAP expression. After stable knockdown of YAP expression using retrovirus, these three groups of TNBC cell lines were assessed for migration, mammosphere formation, and angiogenic properties. Further, detailed characterization of media supernatants was carried out to identify angiogenic markers altered after YAP knockdown. Metastatic potential of the cell lines after YAP knockdown was assessed using mouse tail vein injection model for lung metastasis nodules and lung Mets were analyzed for angiogenic marker. With appropriate ethical approvals, frozen TNBC tumors, along with adjacent normal tissue, were analyzed for YAP and its angiogenic target expression. The expressions were co-related with clinical parameters such as age at diagnosis, grade, lymph node positivity, and recurrence. TNBC cell lines with high YAP and intermediate YAP expression showed significantly reduced migration and mammosphere formation after YAP knockdown. Similarly, media supernatants from high YAP and medium-YAP cell lines showed severely affected angiogenic potential after YAP knockdown. Fluidigm analysis for YAP-signature (Kulkarni et al., Oncotarget 2018) gene expression showed significantly reduced expression of YAP-target genes involved in EMT and stemness in these cell lines. Proteomic analysis of media supernatants revealed expression of two angiogenic factors to be downregulated after YAP knockdown in all 4 cell lines. Metastatic potential of high-YAP expressing cell line in mouse tail vein injection model was severely affected after YAP knockdown. The two TNBC cell lines with low YAP expression did not show any effect on mammosphere formation or angiogenic properties after YAP knockdown; neither had any effect on expression of the angiogenic targets. Patient samples with elevated YAP expression in the tumor compared to adjacent normal tissue showed strong association with younger age at diagnosis, high grade, and lymph node positivity. Our analysis of six TNBC cell lines has uncovered YAP to be significantly associated with increased mesenchymal properties of TNBC cell lines. Analysis of TNBC tissue samples strengthened this association of elevated YAP expression with aggressive clinical parameters within TNBC patients. With further validation, YAP expression can be employed as a prognostic marker to predict aggressive and recurrent TNBC for close follow-up and aggressive treatment planning. Citation Format: Madhura Kulkarni, Nurfarhanah Bte Syed Sulaiman, Supriya Srivastava, Tuan Zea Tan, Thomas Choudary Putti, Xiaomeng Wang, Wanjin Hong, Soo Chin Lee, Yoshiaki Ito. Elevated YAP expression associates with EMT, stemness, and angiogenic properties of TNBC cell lines and recurrence in TNBC patients [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B02.

Pilocytic astrocytomas (PCA) are WHO Grade I tumors with a favorable prognosis. Surgical resection is usually curative. Nonetheless, progressive and/or metastatic disease occurs in 20% of patients. For these patients, treatment options are limited. The role of the immune system in PCA has not previously been reported. We hypothesize that the circulating cytokines contribute to tumorigenicity in PCA. This is an exploratory study with a focus on the identification of circulating cerebrospinal (CSF) cytokines associated with PCA. The primary objective is to demonstrate that CSF cytokines will be differentially expressed in the subset of PCAs that are difficult to treat in comparison to their surgically amendable counterparts. This is a single-institution, retrospective study of prospectively collected data. Patients with a confirmed histological diagnosis of PCA who have simultaneous intraoperative CSF sampling are included. Cerebrospinal fluid samples are subjected to multiplex cytokine profiling. Patient-derived PCA lines from selected patients in the same study cohort are cultured. Their cell culture supernatants are collected and interrogated using the sample multiplex platform as the CSF. A total of 8 patients are recruited. There were two patients with surgically difficult tumors associated with leptomeningeal involvement. Multiplex profiling of the cohort’s CSF samples showed elevated expressions of IFN-γ, IL-2, IL-12p70, IL-1β, IL-4, and TNF-α in these two patients in comparison to the remaining cohort. Next, primary cell lines derived from the same PCA patients demonstrated a similar trend of differential cytokine expression in their cell culture supernatant in vitro. Although our findings are preliminary at this stage, this is the first study in pediatric PCAs that show cytokine expression differences between the two groups of PCA with different clinical behaviors.