Basal-like Breast Cancer Patients Research Articles

Abstract P2-08-12: Development and validation of prognostic gene signatures for basal-like breast cancer and high grade serous ovarian cancer

Abstract Background: Basal-like breast cancer (BLBC) have poor prognosis. Molecular similarities have been reported between BLBC and high grade serous ovarian cancer (HGSOC). To date, there have been no prognostic biomarkers specifically developed for BLBC or HGSOC that can provide risk stratification and inform treatment selections. In this study, we utilized RNA-seq data available from The Cancer Genome Atlas (TCGA) project to develop molecular signatures for risk stratification in BLBC, and further validated these signatures in HGSOC RNA-seq data from TCGA. Methods: Raw count of RNA-seq data were downloaded from TCGA for 190 BLBC and 374 HGSOC patients. The datasets were annotated with 56963 Ensembl gene IDs. Excluding 31375 gene IDs with no greater than 10 counts in at least 90% of the samples, totally 25228 unique Ensembl gene IDs were used. Progression-free interval (PFI) is the primary study endpoint. Analyses of differentially expressed genes were performed using 3 bioconductor packages: DESEq2, edgeR and voom/limma. Signatures based on commonly identified genes among the 3 analytic methods were established using weighted linear combination of gene expression levels. Their performance was evaluated in the BLBC and HGSOC datasets using Kaplan-Meier survival analysis with log-rank tests and Cox proportional hazard regressions. Results: Among 190 TNBC patients, 18 had recurrences within 2 years and 40 showed no recurrences for at least 5 years. These patients were used as recurrent vs. non-recurrent cases for differential expression analysis. 307 and 343 genes were differentially expressed based on adjusted p value threshold 0.05 and 0.01 in DESeq2 and edgeR analysis, respectively. voom/limma identified no genes differentially expressed based on adjusted p values, but 228 genes had unadjusted p values < 0.01 and were used in the following analysis. Taken together, 63, 58 and 21 genes were commonly identified by DESeq2/edgeR, DESeq2/limma and edgeR/limma analysis, respectively. All 3 signatures were able to significantly stratify the TNBC full dataset (n=190) using either 20-, 50- or 80-percentile as the cut-points. When evaluated in HGSOC patients using 80-percentile cut-point, both 63- and 58-gene signatures were able to significantly stratify patients into different risk groups (HR 2.16, 95% CI: 1.4-3.34, p < 0.001; HR 2.06, 95% CI: 1.36-3.11, p < 0.001). Multivariate Cox regression adjusting for age, grade and stage showed 63- and 58-gene signatures remained to be statistically significant in stratifying HGSOC patients (p = 0.0005 and 0.001, respectively). Conclusion: Gene signatures were specifically identified to prognosticate BLBC patients based on RNA-seq data from TCGA project. Which were able to classify HGSOC patients into differential risk groups. With further validations, these signatures may provide additional prognostic tools for clinicians to better manage triple-negative breast cancer that mostly overlap with BLBC, and HGSOC patients who are difficult-to-treat currently. Disclaimer The views expressed in this article are those of the authors and do not reflect the official policy of the department of Army/Navy/Air Force, Department of Defense, or U.S. government. Citation Format: Hu H, Zhang Y, Liu J, Raj-Kumar P-K, Yang H, Lee M, Kovatich AJ, Shriver CD. Development and validation of prognostic gene signatures for basal-like breast cancer and high grade serous ovarian cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-12.

The antipsychotic chlorpromazine suppresses YAP signaling, stemness properties, and drug resistance in breast cancer cells

The major obstacle in current cancer therapy is the existence of cancer stem cells (CSCs), which are responsible for therapeutic resistance and contribute to metastasis and recurrence. Identification of reliable biomarkers for diagnostic and therapeutic targets is necessary for drug development and cancer treatment. In this study, we identified that the antipsychotic chlorpromazine (CPZ) exhibited potent anti-breast cancer and anti-CSC capabilities. Treatment with CPZ suppressed stemness properties including mammosphere formation, aldehyde dehydrogenase (ALDH) activity, and stemness-related gene expressions in breast cancer cells and CSCs. Moreover, CPZ increased the susceptibility of breast cancer MCF7 cells and drug-resistant MCF7/ADR cells when combined with chemotherapies. Mechanistically, we identified that CPZ suppressed yes-associated protein (YAP) through modulating Hippo signaling and promoting proteasomal degradation of YAP. Elevated expression of YAP was confirmed to be crucial for stemness-related gene expressions, and was associated with invasiveness and stem-like signatures in breast cancer patients. Moreover, overexpression of YAP conferred poor outcomes particularly of basal-like breast cancer patients. Our data showed that YAP is a promising therapeutic target for breast CSCs, and CPZ has the potential to be a repurposed drug for breast cancer treatment.

Proteins with Evolutionarily Hypervariable Domains are Associated with Immune Response and Better Survival of Basal-like Breast Cancer Patients

Maltase-glucoamylase (MGAM) and MGAM2 both belong to the glycoside hydrolase family 31. MGAM, a therapeutic target for type 2 diabetes, is α-1,4-glucosidase and expressed in the intestine to catalyze starch digestion. MGAM2, however, is largely uncharacterized. By investigating The Cancer Genome Atlas data, we found that among breast cancer subtypes, MGAM2 expression is nearly exclusive to basal-like breast cancers (BLBCs), whereas MGAM tends to express in luminal A breast cancers. Moreover, MGAM2 expression is associated with better patient survival and correlated with immune genes/signatures, unlike MGAM. Both genes have emerged in mammals, but diverged after the placental-marsupial split. In placentals, MGAM2 has likely lost its α-1,4-glucosidase activity due to mutations in key catalytic sites, and has acquired a large domain that is extracellular, threonine-rich and evolutionarily hypervariable (EHV). Guided by MGAM2 findings, our genome-wide search identified >1000 human proteins with EHV regions. These proteins are enriched in immune functions and molecules, including major histocompatibility complex proteins. Their genes are expressed higher in BLBCs and are associated with better patient survival, like MGAM2. Their EHV-coding sequences are rich in simple repeats and harbor more cancer passenger mutations. In conclusion, MGAM2 diverges from MGAM structurally and likely functionally in placentals. MGAM2 is among >1000 human proteins with EHV regions and associated with immune response. We propose that these EHV molecules may have significant implication in cancer immunotherapy and BLBC treatment.

CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients.

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple-negative breast cancer (TNBC). In a mass spectrometry-based global proteomic study of 44 formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal-like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence-free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1-2.85), and this correlation was recapitulated in a cohort of 580 basal-like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06-1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20-6.19), particularly those who were lymph node tumor-negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07-4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance.

ADAR1-mediated RNA-editing of 3\u2032UTRs in breast cancer

BackgroundWhole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA (ADAR) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed.ResultsWe characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3′UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3′UTRs. Interestingly, editing was particularly increased in the 3′UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1).ConclusionsAltogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.

An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer.

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.

MiR-424-5p regulates cell proliferation, migration and invasion by targeting doublecortin-like kinase 1 in basal-like breast cancer

Our previous study has showed doublecortin like kinase 1 (DCLK1) serves as an oncogene to regulate basal-like breast cancer cell proliferation, migration and invasion, and is associated with malignant status and poor prognosis. The aim of this study is to identify microRNAs (miRNAs), which target DCLK1 to regulate basal-like breast cancer cell proliferation, migration and invasion. In our results, we observed that miR-424-5p expression was decreased in basal-like breast cancer tissues and cell lines. Furthermore, we found 3’-UTR of DCLK1 had binding site of miR-424-5p based on microRNA target databases, and there was an inverse correlation between miR-424-5p and DCLK1 in basal-like breast cancer tissues. Moreover, we confirmed miR-424-5p directly targeted to 3’-UTR of DCLK1 through luciferase reporter assay, and miR-424-5p negatively regulated DCLK1 mRNA and protein expressions through qRT-PCR and western blot. The gain-of-function studies showed that miR-424-5p suppressed basal-like breast cancer cell proliferation, migration and invasion. The rescued-function studies suggested up-regulation of DCLK1 could rescue inhibition of miR-424-5p mimics in the regulation of basal-like breast cancer cell proliferation, migration and invasion. Finally, low-expression of miR-424-5p was associated with advanced clinical stage, large tumor size, more metastatic lymph nodes, present distant metastasis and poor histological grade in basal-like breast cancer patients. In conclusion, miR-424-5p is a tumor suppressive microRNA to regulate tumor cell proliferation, migration and invasion via binding to the functional target DCLK1, and associated with malignant status in basal-like breast cancer.

FOXF2 deficiency permits basal-like breast cancer cells to form lymphangiogenic mimicry by enhancing the response of VEGF-C/VEGFR3 signaling pathway

Lymphatic metastasis is the main route of breast cancer metastasis. It is known that lymphangiogenesis facilitates lymphatic metastasis through vascular endothelial growth factor-C (VEGF-C)/VEGF receptor 3 (VEGFR3) pathway-linked interactions between the tumor and its microenvironment. Here, we report a novel mechanism of lymphatic metastasis by which aggressive basal-like breast cancer (BLBC) cells form lymphatic vessel-like structures that are identified by the positive expression of lymphatic endothelial cell markers lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), podoplanin, and VEGFR3, and termed as lymphangiogenic mimicry (LM), for the first time. Our clinical evidence and experimental data in vivo and in vitro revealed that forkhead box F2 (FOXF2) deficiency promotes the lymphatic metastasis of BLBC by conferring a lymphangiogenic mimetic feature upon cancer cells through directly activating VEGFR3 transcription. The fact that FOXF2 controls the activation of the VEGF-C/VEGFR3 signaling pathway in BLBC cells provides potential molecular diagnostic and therapeutic strategies for lymphatic metastasis in BLBC patients.

The suppression of DUSP5 expression correlates with paclitaxel resistance and poor prognosis in basal-like breast cancer.

Basal-like breast cancer (BLBC) is resistant to endocrinotherapy and targeted therapy and new molecular therapies are needed for BLBC. In this study, we evaluated the role of DUSP1 and DUSP5, negative regulators of mitogen-activated protein kinase pathway, in the aggressiveness of BLBC. MDA-MB-231 cells were given paclitaxel (PTX) treatment and subsequently PTX resistant cell clones were established. Microarray analysis, real-time quantitative reverse transcription PCR (qRT-PCR), and online analysis of large cohorts of breast cancer patients were performed. The PTX resistant cells showed stronger cell proliferation ability by exhibiting the upregulation of CENPF, CDC6, MCM3, CLSPN and SMC1A expression. Furthermore, DUSP1 and DUSP5 expression was significantly downregulated in PTX resistant cells. In addition, in large breast cancer patients' database, both DUSP1 and DUSP5 correlated negatively with higher histological grade. DUSP1 low expression was obvious in HER2 positive and basal like while DUSP5 low expression was peculiar for basal like compared with other subtypes. Remarkably, low expression of DUSP5, but not DUSP1, was significantly correlated with poor survival of BLBC patients. In conclusion, our data suggest that loss of DUSP5 expression results in PTX resistance and tumor progression, providing a rationale for a therapeutic agent that restores DUSP5 in BLBC.

Stromal Infiltration of Tumor-Associated Macrophages Conferring Poor Prognosis of Patients with Basal-Like Breast Carcinoma.

Aims: Tumor associated macrophages (TAMs) play a critical role in the initiation and progression of breast cancer. However, their prognostic significance in the molecular subtype of basal-like breast cancer (BLBC) is poorly understood. The aim of this study was to investigate the extent and patterns of TAMs in BLBC and their associations with clinicopathological features and patient survival.Methods and Results: We evaluated TAMs in 200 cases of BLBC by immunohistochemistry using the M2 macrophage marker CD163 and the pan-macrophage marker CD68 in tumor nest and stroma, and assessed their prognostic significance.The study demonstrated that infiltration of CD163+ and CD68+ macrophages in tumor stroma was of clinical relevance in BLBC, but not those in tumor nest. Increased stromal infiltration of CD68+ or CD163+ macrophages correlated with larger tumor size, higher histological grade, higher 5-year recurrence and 5-year breast cancer mortality. Although both of CD68+ and CD163+ macrophages in tumor stroma were associated with poor recurrence-free survival (RFS) and overall survival (OS), multivariate analysis demonstrated that only CD163+ macrophage was an independent predictor of RFS and OS.Conclusions: Our results highlight the prognostic importance of TAMs' location in BLBC. CD163, a highly specific biomarker for M2 macrophages, is an independent prognostic marker for BLBC patients, and may serve as an indicator or potential target of macrophage-centred therapeutic strategies.

Ubiquitin-conjugating enzyme E2T (UBE2T) and denticleless protein homolog (DTL) are linked to poor outcome in breast and lung cancers

Protein ubiquitination and degradation represent druggable vulnerabilities of cancer cells. We used gene expression and functional annotation analyses to identify genes in the ubiquitin pathway which are differentially expressed between normal breast and basal-like tumors. With this approach we identified 16 ubiquitin related genes overexpressed in basal-like breast cancers compared with normal breast. We then explored the association between these genes and outcomes using the KMPlotter online tool. Two genes, the ubiquitin-conjugating enzyme E2T (UBE2T) and the denticleless protein homolog (DTL) were overexpressed and linked with detrimental outcome in basal-like and luminal breast cancer patients. Furthermore, we found that UBE2T and DTL were amplified in around 12% of breast tumors based on data contained at cBioportal. In non-small cell lung adenocarcinomas, UBE2T and DTL were also amplified in around 7% of cases and linked with disease recurrence after surgical resection. No significant molecular alterations or a clear trend for clinical outcome was observed for these genes in ovarian serous cystadenocarcinoma, esophagus-stomach cancer or non-small squamous cell carcinoma. Our data suggest that UBE2T and DTL may have a role in the pathophysiology of breast and lung tumors, opening avenues for future clinical evaluation of agents targeting those proteins or their pathways.

Abstract 5020: A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer

Abstract Cells residing in mesenchymal state are often associated with stem cell properties. The phenotypic changes from epithelial to mesenchymal cell state, or from non-stem-like to stem-like cell state contribute to tumor heterogeneity and play important roles in tumor initiation, progression and metastasis. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen to identify regulators of mesenchymal and stem-like cell states using a barcoded human ORFome expression library in human mammary epithelial cells. In the screen, we used flow cytometric analysis of the CD44 cell surface marker and identified 68 ORFs that can switch cells from CD44 low state to CD44 high state in 7 days. Among these genes, the RNA splicing factors were highly enriched as analyzed by GO terms and Gene set enrichment analysis (GSEA).We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. We discovered that QKI and RBFOX1 were both necessary and sufficient to promote EMT and stem-like states. MBNL1, MBNL2 and CELF4 were sufficient to induce some mesenchymal markers. We further investigated the downstream targets of these splicing factors by RNA-sequencing analysis. We found that QKI and RBFOX1 regulated the alternative splicing of genes in 5 functional modules: 1) Cell motility and ECM/cytoskeleton organization; 2) Stem cell fate determination; 3) Oncogenic signaling; 4) Epigenetic targets; 5) cell polarity. Strikingly, using molecular and biochemical assays, we found that QKI and RBFOX1/2 interacted and cooperatively regulated the alternative splicing of a large number of overlapping transcripts, including Filamin B (FLNB). QKI and RBFOX1 induced a shorter isoform via exon skipping. which plays a key functional role in the regulation of EMT. Importantly, the expression of QKI, RBFOX1/2 and the short isoform of FLNB are elevated in basal B type of breast cancer cell lines and in basal-like breast cancer patient samples, the subtype of breast cancer that displays higher degree of mesenchymal and stem-like traits. In conclusion, alternative RNA splicing plays a key role in the regulation of EMT and stem-like cell states. QKI and RBFOX1/2 are both necessary and sufficient to promote EMT and stem-like traits. Alternative splicing of FLNB controlled by QKI and RBFOX1/2 is one of the key downstream targets that regulates EMT. Thus, the molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5020. doi:10.1158/1538-7445.AM2017-5020

Dual function of MAZ mediated by FOXF2 in basal-like breast cancer: Promotion of proliferation and suppression of progression

Myc-associated zinc finger protein (MAZ) is a transcription factor with C2H2-type zinc-finger motifs that can bind GC-rich cis-elements. MAZ activates the transcription of some cancer-related genes and represses that of others, suggesting that changes in MAZ expression may play different roles in the development and progression of different types or subtypes of cancers depending on its target genes. However, the functions and mechanisms of MAZ in regulating the carcinogenesis and progression of breast cancer have remained unclear. In the current study, we show that MAZ performs dual function in basal-like breast cancer (BLBC): suppression of aggressiveness and promotion of proliferation. Forkhead box F2 (FOXF2) is a novel transcription target of MAZ and mediates the functions of MAZ. The MAZ mRNA level, particularly in combination with the FOXF2 mRNA level, may serve as a prognostic marker for BLBC patients. Our results indicate that the dual function of the MAZ-FOXF2 axis reflect the pleiotropic nature of multifunctional transcription factors in regulating the different stages of cancer development and progression, which could lead to the complexity of cancer diagnosis and treatment.

Science Signaling Podcast for 21 February 2017: Pentraxin-3 in basal-like breast cancer.

This Podcast features an interview with Antoine Karnoub, senior author of a Research Article that appears in the 21 February 2017 issue of Science Signaling, about how pentraxin-3 (PTX3) links increased phosphoinositide 3-kinase (PI3K) signaling to stem cell properties in basal-like breast cancer (BLBC). BLBC is an aggressive type of breast cancer in which the tumor cells exhibit increased PI3K signaling and have stem cell-like properties. Thomas et al found that aberrant PI3K signaling in BLBCs stimulated the expression of PTX3, which encodes a protein that functions in innate immunity. PTX3 promoted stem cell-like traits in mammary epithelial cells and stimulated the growth of breast cancer cells. Conversely, decreasing the abundance of PTX3 in breast cancer cells reduced both the growth of these cells and their expression of stem cell markers. The abundance of PTX3 transcripts in breast tumors negatively correlated with patient survival, suggesting that PTX3 may be a useful biomarker for stratifying BLBC patients and that targeting PTX3 may suppress tumor growth in some BLBC patients.Listen to Podcast.

Abstract P1-08-05: Discovering drug targets for aggressive breast cancer with TP53 missense mutations by a genome-wide screen

Abstract Breast cancer is a heterogeneous disease and has subtypes with distinct phenotypic and molecular characteristics. Genetically, 30% of all breast tumors and 80% of the basal-like breast cancer (BLBC) subtype harbor mutations in TP53 and numerous sporadic somatic mutations in other genes. This molecular heterogeneity has posed a challenge in developing safe and effective therapeutic regimens to treat a broad spectrum of breast cancer patients, and discovery of intra-subtype along with inter-subtype heterogeneity has added another layer of complexity for targeted therapies. Based on the analysis of somatic mutation profiles in the BLBC subtype, we identified a wide variety of TP53 missense mutations and thousands of co-existing mutations, which led us to hypothesize that intra-subtype heterogeneity is derived from combinatorial effects of neo-morphic (gain-of-function) activities of different types of missense mutant p53 proteins and complex interplay between specific driver mutations in TP53 and a distinct subset of functionally important co-mutations (or “co-drivers”). We ectopically overexpressed the ten most prevalent missense mutations in TP53 found in breast cancer tumors in non-transformed mammary epithelial cells and examined their cellular functions associated with the hallmarks of cancer. The results showed widely different spectrums of phenotypic changes in cell proliferation, resistance to apoptosis, cell invasion, anoikis resistance and cell polarity. As a proof of concept for the 'co-drivers', we knocked-out PTEN in a non-invasive TP53 mutant cell using the CRISPR and shRNA systems. Inactivation of PTEN in non-invasive TP53-mutant cell increased its invasiveness. TP53 mutations and PTEN deletions are frequent in BLBC patients, who have significantly higher rates of metastasis, recurrence and a lower survival rate than the other subtypes. The absence of three signaling hormone receptors, which can be targeted by specific inhibitors, significantly limits available therapies for the majority of BLBC patients to surgery and cytotoxic chemo/radiotherapies. Combination therapies targeting the driver and co-driver gene associated pathways could be quite promising solutions to treat aggressive breast cancer cases. To address this, we have developed a genome-wide CRISPR based gene knock-out screening approach to test the effect of loss-of-function of individual human genes in clinically important TP53 mutant backgrounds that can promote cancer-like behaviors and identify the combinations of 'TP53 mutation and co-driver pathways' that can be targeted by specific inhibitors. Citation Format: Pal A, Park J, Gonzalez-Malerva L, Eaton S, LaBaer J. Discovering drug targets for aggressive breast cancer with TP53 missense mutations by a genome-wide screen [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-05.

Doublecortin-like kinase 1 is a novel biomarker for prognosis and regulates growth and metastasis in basal-like breast cancer

Doublecortin like kinase 1 (DCLK1) was observed overexpressed in several types of human cancers, and played a functional role in tumorigenicity, but little is known about the biological function of DCLK1 in basal-like breast cancer. In our results, the expressions of DCLK1 mRNA and protein were elevated in basal-like breast cancer tissues and cell lines. DCLK1 high-expression associated with clinical stage, lymph node metastasis, distant metastasis and histological grade. DCLK1 high-expression was an independent unfavorable prognostic factor for basal-like breast cancer patients. The biological experiments suggested knockdown of DCLK1 induced inhibition of basal-like breast cancer cells proliferation, migration and invasion. In conclusion, DCLK1 serves as a prognostic biomarker for basal-like breast cancer and a potential therapeutic target for basal-like breast cancer.

HMMR antisense RNA 1, a novel long noncoding RNA, regulates the progression of basal-like breast cancer cells.

Recently, accumulating evidence has suggested that long noncoding RNAs (lncRNAs) play crucial roles in carcinogenesis and cancer progression. Hyaluronan-mediated motility receptor (HMMR) is an essential cancer-related gene in basal-like breast cancer (BLBC). In our study, HMMR antisense RNA 1 (HMMR-AS1) was analyzed in BLBC patients through polymerase chain reaction analysis. Here, we found that the expression of HMMR was positively correlated with HMMR-AS1 (RP11-80G.1). When HMMR-AS1 (RP11-80G.1) was knocked down, the expression of HMMR markedly reduced. Furthermore, in MDA-MB-231 and MDA-MB-468 breast cancer cells, the proliferation and migration abilities were remarkably suppressed via knocking down HMMR-AS1 (RP11-80G.1) in vitro. The results showed that lncRNA HMMR-AS1 (RP11-80G.1) influenced the progression of BLBCs through regulating HMMR, suggesting that HMMR-AS1 (RP11-80G.1) could be regarded as a novel biomarker and therapeutic target in the treatment of BLBCs in future.

FEATURES OF LYMPHOCYTES IN THE BLOOD IN PATIENTS WITH BASAL-LIKE BREAST CANCER SOUTH-EAST UKRAINE

Lymphocytes are important in antitumor immunity. Therefore, the estimate of the magnitude of lymphocytes, could be used to determine the protective capabilities of the organism basal-like breast cancer patients. The purpose of the study: building a morphological features peripheral blood lymphocytes of patients with basal-like breast cancer South-East Ukraine, receiving comprehensive chemoradiotherapy. It was found that treatment BLT changes the structure of peripheral blood lymphocytes. After the comprehensive treatment of peripheral blood lymphocytes of patients significantly increased in size, they decreased nuclear-cytoplasmic ratio. For definitions immune status of patients basal-like breast cancer no small role played by morphological characteristics of lymphocytes in the peripheral blood before and after complex treatment.

Abstract P5-02-04: Plasma autoantibodies associated with basal-like breast cancers

Abstract Background: Basal-like breast cancer (BLBC) is a rare aggressive subtype that is less likely to be detected through mammographic screening. Identification of circulating markers associated with BLBC could have promise in detecting and managing this deadly disease. Methods: Using samples from the Polish Breast Cancer study, a high-quality population-based case-control study of breast cancer, we screened 10,000 antigens on protein arrays using 45 BLBC patients and 45 controls, and identified 748 promising plasma autoantibodies (AAbs) associated with BLBC. ELISA assays of promising markers were performed on a total of 145 BLBC cases and 145 age-matched controls. Sensitivities at 98% specificity were calculated and a BLBC classifier was constructed. Results: We identified a 13-AAbs (CTAG1B, CTAG2, TP53, RNF216, PPHLN1, PIP4K2C, ZBTB16, TAS2R8, WBP2NL, DOK2, PSRC1, MN1, TRIM21) that distinguished BLBC from controls with 33% sensitivity and 98% specificity. We also discovered a strong association of TP53 AAb with its protein expression (p=0.009) in BLBC patients. In addition, MN1 and TP53 AAbs were associated with worse survival (MN1 AAb marker HR=2.25 95%CI= 1.03-4.91 p=0.04; TP53, HR=2.02, 95%CI 1.06-3.85, p=0.03). We found limited evidence that AAb levels differed by demographic characteristics. Conclusions: These AAbs warrant further investigation in clinical studies to determine their value for further understanding the biology of BLBC and possible detection. Currently, they are also being tested in a large national blind validation trial using a well characterized independent sample set. Citation Format: Wang (Student) J, Figueroa JD, Wallstrom G, Barker K, Park JG, Demirkan G, Lissowska J, Anderson K, Qiu J, LaBaer J. Plasma autoantibodies associated with basal-like breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-02-04.

CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis.

A critical process underlying cancer metastasis is the acquisition by tumor cells of an invasive phenotype. At the subcellular level, invasion is facilitated by actin-rich protrusions termed invadopodia, which direct extracellular matrix (ECM) degradation. Here, we report the identification of a new cytoskeletal component of breast cancer cell invadopodia, namely cysteine-rich protein 2 (CRP2). We found that CRP2 was not or only weakly expressed in epithelial breast cancer cells whereas it was up-regulated in mesenchymal/invasive breast cancer cells. In addition, high expression of the CRP2 encoding gene CSRP2 was associated with significantly increased risk of metastasis in basal-like breast cancer patients. CRP2 knockdown significantly reduced the invasive potential of aggressive breast cancer cells, whereas it did not impair 2D cell migration. In keeping with this, CRP2-depleted breast cancer cells exhibited a reduced capacity to promote ECM degradation, and to secrete and express MMP-9, a matrix metalloproteinase repeatedly associated with cancer progression and metastasis. In turn, ectopic expression of CRP2 in weakly invasive cells was sufficient to stimulate cell invasion. Both GFP-fused and endogenous CRP2 localized to the extended actin core of invadopodia, a structure primarily made of actin bundles. Purified recombinant CRP2 autonomously crosslinked actin filaments into thick bundles, suggesting that CRP2 contributes to the formation/maintenance of the actin core. Finally, CRP2 depletion significantly reduced the incidence of lung metastatic lesions in two xenograft mouse models of breast cancer. Collectively, our data identify CRP2 as a new cytoskeletal component of invadopodia that critically promotes breast cancer cell invasion and metastasis.