Enhanced immortalization, HUWE1 mutations and other biological drivers of breast invasive carcinoma in Black/African American patients

BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels. METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models. RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P < 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P < 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone. CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC. Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-15.

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Background: Breast cancer constitutes 30% of all new cancer cases in women. Despite steady decrease in breast cancer mortality, the lack of therapeutic targets is still a major problem. While the hormone-receptor positive (ER+) and human epidermal growth factor receptor 2-positive (HER2+) breast cancers respond to current targeted therapies, no targeted therapy is available for the treatment of triple negative breast cancer (TNBC), which lacks the expression of ER alpha (ER), progesterone receptor (PR), and HER2-receptor. Hence, there is an urgent need for effective targets against this sub-type of breast cancer. Previous studies in our laboratory used gene expression profiling of human breast cancers to identify kinases overexpressed in ER-negative breast cancers. One of these highly expressed kinases is the maternal embryonic leucine-zipper kinase (MELK). MELK is a serine/threonine protein kinase known to have a role in cell cycle progression, apoptosis and DNA repair. The purpose of this study was to test the hypothesis that MELK is required for the growth and migration of TNBC. Methods: RNA and protein was isolated from a panel of TNBC and ER-positive breast cancer cell lines and MELK expression was quantified by qPCR and immunoblotting. To determine whether MELK regulates cell growth, ER-positive and TNBC cell lines were transfected with siRNA targeting MELK and cell number was measured by manual counting. Anchorage-independent growth was measured using soft agar assays. Effect on migration and invasion was determined using Boyden Chamber assays. Immunostaining with actin-phalloidin was performed on MELK knockdown cells to determine effect of MELK loss on the cytoskeleton. Results: MELK mRNA and protein levels were significantly higher in TNBC cell lines compared to ER-positive breast cancers. Knockdown of MELK suppressed growth (≥50% growth inhibition) in six TNBC cell lines but had no effect on growth of six ER positive cell lines. Colony formation was also greatly reduced in TNBC cell lines but was not affected in ER positive cell lines upon siRNA knockdown of MELK. In addition, knockdown of MELK reduced migration of three TNBC cell lines (MDAMB231, MDAMB468 and Hcc70) but had no effect on the ER-positive cell line (MCF7). Decreased staining of actin filaments was observed in cell lines where migration was reduced upon MELK knockdown suggesting a role of MELK in formation of actin cytoskeleton. Current studies are focused on understanding how MELK regulates the cell growth and migration in TNBC. Conclusions: MELK is an important growth regulator of TNBC, but not of ER positive breast cancers. Our results indicate that MELK promotes cell migration in TNBC cells. These findings suggest that MELK is a promising target for the treatment of TNBC. Supported by a Susan G. Komen for the Cure Promise Grant (KG081694), and the John Charles Cain Award. Citation Format: Nidhi Batra, Corey Speers, Ivan Uray, Abhijit Mazumdar, Anna Tsimelzon, Susan Hilsenbeck, Gordon Mills, Powel Brown. Maternal embryonic leucine zipper kinase is critical for the growth and migration of triple negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2014-3299

Introduction: several retrospective studies suggest that ßAR blocking drugs (BB) are associated with improved survival in patients with a wide range of cancers. Recently, we retrospectively showed an association between BB intake and improved progression free survival in patients with HER2 negative advanced breast cancer (BC), particularly striking in triple-negative disease (TNBC) (Reference). Based on this finding we decided to conduct an in silico study in which we have interrogated ßARs in a publicly available BC sample database and the Translational Oncology Research Lab (TORL) translational platform with a genomic, transcriptomic and proteomic approach. Methodology: genomic and transcriptomic data sets for ßAR 1, 2 and 3 were retrieved from cBioPortal considering all BC samples available with this information in The Cancer Genome Atlas (TCGA). Transcriptomic and proteomic data sets from 48 BC cell lines obtained from TORL were queried for ßARs as well and used with validation and exploratory intent. Mutations, amplifications and deletions were queried in DNA; gene expression profiles were interrogated using RNAseq data together with protein expression by RPPA. Average expression, log ratio and fold change in mRNA and Reverse Phase Protein Array (RPPA) quantitative assessments for corresponding proteins were noted. BC cell lines with top 10 mRNA and protein levels of ßAR 1, 2 and 3 were identified. Results: CBioPortal DNA data shows ß AR1 amplified in 1-10% and deleted in 0,4%; ß-AR2 amplified in 1-3% and deleted in 0,1%; ß-AR3 amplified in 15-20% and deleted in 2% of the BC samples. CBioPortal mRNA data shows ß-AR1 is upregulated in 2.7% (mostly Progesterone Receptor negative BC); ßAR2 is upregulated in 4% (mostly TNBC); ßAR3 is upregulated in 4% (mostly HER2 negative BC) in BC samples. TORL cell line panel shows that ßARs are heterogeneously expressed between the BC cell lines (fold change range: ßAR 1 2.015-3.636; ßAR2 2.545-8.248; ßAR3 1.809-2.444). Within the 10 BC cell lines with highest ßAR1 and ßAR2 expression, 7(COLO-824, HCC1937, BT-549, BT-20, HCC1599, HCC1143, HCC1806) and 6 (184A1, MDA-MB-231, 184B5, HCC1806, MCF-10A and MDA-MB-468) of them respectively correspond to the basal BC subtype. RPPA identifies caveolin 1, PAI 1, EGFR and Bax as the proteins with the higher co-expression with ßAR1 and ßAR2. Conclusions: DNA alterations are infrequent in ßARs in BC samples. Transcriptional mRNA data from BC samples shows ßARs mostly expressed in non-luminal BC subtypes, being ßAR 2 the one with highest expression. In silico data results from BC cell line panel show ßAR 1 and ßAR 2 are highly expressed in basal BC subtype. The above data suggest that ßAR, and ßAR 2 in particular could be a relevant target to explore in in vivo BC models. Table 1:mRNA and RPPA in BC cell linesCell lineAverage mRNALog RatioFold ChangeCaveolin 1PAI1EGFR184A1313173.048.244.183.411.72SUM-190223292.811.03-1.37-0.570.09MDA-MB-231151002.997.993.853.251.57184B5130121.973.933.902.361.82HCC1806124112.375.193.750.491.67MCF-10A119122.194.583.892.670.08ZR-75-3093001.893.72-1.51-0.61-0.25JIMT-163541.593.013.082.010.81MDA-MB-41562391.242.370.96-0.39-0.13MDA-MB-46862801.342.541.06-0.251.72 Citation Format: Spera G, Von Euw E, Fresco R, Slamon DJ. Backwards translation: Exploring beta-adrenoreceptors (ßAR) in triple negative breast cancer (TNBC), a novel and druggable pathway [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 P2-03-07.

Breast cancer is a major cause of cancer related death in United States women, and there is an urgent need for novel targeted therapies with low toxicity and increased efficacy. The TRAIL pathway has been of interest in the field of oncology, but resistance has been identified in cancer cell lines and primary tumors including those of the breast. Only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL, with other breast cancer molecular subtypes being resistant (Rahman et al., Breast Cancer Res. Treat., 2008). Clinical translation of these findings has been limited, as TRAIL receptor agonistic antibodies have shown minimal effects. We identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10 (Allen et al., Sci. Trans. Med., 2013) that functions through dual inhibition of AKT/ERK, induction of the ATF4/CHOP pathway (Kline et al., Submitted, 2015) and cytotoxic effects dependent on TRAIL/DR5 upregulation. Recently, ONC201 completed its first-in-man phase 1 clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-EORTC meeting). The objective of this work was to investigate ONC201 efficacy in TNBC (TRAIL-sensitive) and non-TNBC (TRAIL-resistant) cells. Using cell viability assays, we demonstrate IC50 values for ONC201 in the low micromolar range for both TNBC (n = 6) and non-TNBC cells (n = 5). These doses are achievable based on human PK. The evaluated non-TNBC cell lines, including those with estrogen receptor positivity and HER2 amplifications, have been shown to be highly resistant to TRAIL therapy. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in both TNBC and non-TNBC cells. We also show that a single dose of ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 triple negative human breast cancer xenograft model. The effects of ONC201 on well-known mechanisms of TRAIL-resistance, such as increases in inhibitor of apoptosis (IAP) family proteins, were investigated to explain sensitivity of TRAIL-resistant breast cancer cells to ONC201. We observed that ONC201 mediates a decrease in expression of IAP family proteins XIAP, c-IAP1, and c-IAP2 across TNBC and non-TNBC cell lines. We have shown that XIAP levels correlate with sensitivity to ONC201-induced apoptosis (Kline et al., Submitted, 2015). We also examined levels of death receptors DR4 and DR5 using flow cytometry, as a known mechanism of TRAIL resistance involves constitutive death receptor endocytosis from the cell surface (Zhang et al., Mol. Cancer Res., 2008). Interestingly, increases in cell surface DR4 and DR5 levels are greater in TNBC cell lines when compared with non-TNBC cell lines. Overall, our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC and non-TNBC subtypes. These effects are observed regardless of the TRAIL sensitivity of the cells, and may be mediated through mechanisms involving downregulation of anti-apoptotic proteins and upregulation of cell surface death receptors. Our work will further understanding of these mechanisms and contribute to development of a preclinical rationale for the use of ONC201 as a treatment for breast cancers. Citation Format: Marie D. Baumeister, Jessica Wagner, Christina L.B. Kline, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A16.

Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P < 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P < 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Introduction: Diagnosis of triple negative breast cancer (TNBC) is associated with adverse prognosis particularly in case of chemotherapy resistance. TNBC is a heterogeneous entity and seems to consist of at least six distinct molecular subtypes (Lehman subtypes) with distinct chemotherapy sensitivity. The cytotoxic agent eribulin induces tumor cell apoptosis through depolymerization of the cell spindle apparatus. Based on clinical data it has recently been suggested that TNBC is particularly sensitive against eribulin. The goal of this analysis was to compare (i) TNBC vs. non TNBC lines and (ii) cell lines of distinct TNBC subtypes with regard to eribulin sensitivity in vitro. Methods: 17 established breast cancer cell lines comprising both TNBC (4 basal-like 1/2; 1 mesenchymal; 3 mesenchymal stem cell; 1 interleukin; 2 luminal AR; 1 unclassified) and non-TNBC (n=5) phenotypes were cultured and subjected to cell viability assay (MTT test), migration experiment (scratch assay), apoptosis analysis (Western Blot experiment for PARP cleavage) and quantitative RT-PCR analysis (for GABRP gene expression) after exposure to eribulin or control. Furthermore, gene expression of 8 genes known to induce malignant transformation (MMP7, ELF5, YBX1, RARRES1, PRNP, SOX 10, EGFR and GABRP) was analyzed via quantitative RT-PCR analysis in the triple negative cell line MDA-MB 231 after exposure to eribulin or control. Results: The effect of eribulin on the cell viability varied to a lesser extent among the TNBC compared to the non-TNBC cell lines though we could not observe a significant difference between both groups. Mentionable the TNBC cell line DU 4475 representing the interleukin phenotype displayed a significant stronger resistance to eribulin compared to all other phenotypes. A decelerated migration could be observed in the TNBC cell line MDA-MB 231 after exposure to the IC50 concentration of eribulin compared to non-treated cells. Induction of apoptosis by eribulin treatment was verified by PARP cleavage in various TNBC cell lines. GABRP known to be overexpressed especially in basal like TNBC showed a slight increase in gene expression after exposure to eribulin in various phenotypes of TNBC - most prominent in MDA-MB 231. Additionally, upregulation of ELF5 and downregulation of YBX1 and PRNP, and, to a lesser extent, of MMP7 and SOX 10 gene expression could be investigated in MDA-MB 231 after eribulin treatment. Conclusion: We did not observe a significant association with regard to eribulin sensitivity between TNBC and non-TNBC. Chemotherapy sensitivity varied to a lesser extent among TNBC cell lines compared to non-TNBC cell lines. Eribulin inhibits cell proliferation and migration, induces apoptosis in TNBC, and influences gene expression of overexpressed genes in TNBC known to participate in and induce malignant transformation. Though the current work did not explicitly specify one phenotype of TNBC for eribulin treatment regarding chemotherapy sensitivity, we identified possible target genes influenced by eribulin treatment, e. g. GABRP, and therefore need further investigation for a potential treatment approach combining eribulin with e. g. GABRP inhibitor. Citation Format: Bräutigam K, Mitzlaff K, Uebel L, Steinert G, Köster F, Polack S, Rody A, Liedtke C. Association between phenotype of triple negative breast cancer cell lines and sensitivity against eribulin mesylate in vitro. [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 P6-08-07.

Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer (TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression. Key words: Triple negative breast neoplasms; microRNAs; Membrane proteins; Phosphoric monoester hydrolases; Neoplasm metastasis

Exploring the Potential of Breast Microbiota as Biomarker for Breast Cancer and Therapeutic Response

Background: Cyclin-dependent-kinase-4/6 inhibitors (CDK4/6is) plus endocrine therapy (ET) are standard of care first-line treatment for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (mBC). However, the emergence of resistance to CDK4/6is plus ET presents a clinical challenge with few treatment alternatives. The effectiveness of CDK4/6is in patients with triple-negative (TN) breast cancer remains uncertain, although the potential synergy with other targeted therapies is currently under investigation. Our study demonstrates the enhanced and synergistic activity of BLU-222, a selective CDK2 inhibitor, when combined with CDK4/6is in preclinical models of both HR+/HER2- and TN breast cancer resistant to CDK4/6is. Methods: Palbociclib resistant (PR) HR+/HER2- (MCF7 and T47D) and TN (MDA-MB-231 and BT-20) breast cancer cell lines were generated by escalating palbociclib concentrations in culture. Using SynergyFinder, we assessed the effect of BLU-222 alone and in combination with palbociclib in the highest single-agent model in vitro. The effectiveness of BLU-222, alone or in combination with palbociclib, was further evaluated in four patient-derived xenograft (PDX) models from HR+/HER2- patients whose tumors progressed after palbociclib plus ET, two TN PDX models, and a TN breast cancer transgenic model driven by tumor-specific forms of cyclin E. Results: The PR HR+/HER2- and TN breast cancer cell lines, unresponsive to palbociclib, exhibited significantly increased sensitivity to BLU-222. The combination of BLU-222 and palbociclib in all four cell lines revealed a robust synergistic effect in PR cells, inducing enhanced apoptosis and cell cycle alterations in G1 or G2/M phases. Treatment with BLU-222 and palbociclib demonstrated substantial antitumor activity in all six PDX models and the cyclin E high TN transgenic models, surpassing the effects of individual treatments in each model. This combination led to lasting tumor regression and extended survival, even after treatment discontinuation. Mechanistically, treatment with BLU-222, alone or combined with palbociclib, induced the expression of p21 and/or p27 in all in vivo models, which we hypothesize sensitizes tumors to palbociclib. In vitro, the CRISPR knockout of p21 or p27 in MCF-7 PR cells abolished the synergistic activity of BLU-222 and palbociclib, confirming the crucial involvement of p21 and p27 in influencing the treatment's effectiveness. Conclusions: We found robust activity of the CDK2 inhibitor, BLU-222 when combined with CDK4/6is in resistant HR+/HER2- and TN breast cancer cell lines and in vivo models. These results support the potential clinical utility of BLU-222 in combination with CDK4/6is for the treatment of both subtypes of breast cancer. Citation Format: Linjie Luo, Yan Wang, Tuyen Bui, Mi Li, Serena Kim, Juliana Navarro-Yepes, Nicole M. Kettner, Debasish Tripathy, Kelly K. Hunt, Kerrie Faia, Khandan Keyomarsi. Anti-tumor activity of CDK2 inhibitor BLU-222 in combination with CDK4/6 inhibitors for overcoming resistance in HR positive and triple negative metastatic breast cancers models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4622.

Aim. To investigate the possibility of using radiation diagnostic data to determine various molecular subtypes of breast cancer (BC) using artificial intelligence technologies.Materials and methods. The material for the study was retrospective data of 344 patients treated at the Sverdlovsk Regional Oncology Dispensary in the period from 2021 to 2023. The average age of the study sample was 56.8 ± 10.6 years, ranging from 33 to 82 years. All patients were diagnosed with BC, confirmed histologically. Molecular subtypes of BC were assessed based on trepan biopsy and surgical material. All patients underwent mammographic, ultrasound, and magnetic resonance imaging examinations, and sets of diagnostic features were identified that most accurately correspond to various molecular subtypes of BC. To achieve this goal, the authors identified the following diagnostic features: age, maximum diameter of the formation measured for various methods of radiation diagnostics, morphological features (contours, spatial orientation, shape of the detected formations or areas of reconstruction, heterogeneity of the structure of formations, presence of calcifications, characteristics of blood flow in the tumor) and dynamic parameters of paramagnetic accumulation during magnetic resonance imaging of the mammary gland.Based on the histological examination data, the degree of tumor differentiation (G), proliferative activity index (Ki-67), regional lymph node status (presence or absence of metastases), and molecular-immunohistochemical tumor subtype were assessed. An analysis was conducted to determine whether there was a statistically significant relationship between diagnostic features and molecular subtypes of BC. The analysis was performed by conducting chi-square tests for features and subtypes (classes) of BC, previously converted to binary form. From the arrays of values s elected for the study of diagnostic features, training and test samples were formed, and an algorithm for the classification model of artificial intelligence was determined. The accuracy of BC typing was ensured by using a combination of 7 diagnostic features and 6 classification models: five single-class and one multi-class. The gradient boosting algorithm (GradientBoostingRegressor) was used to train single-class models. The strategy “one (class) versus the rest” was used to train the multi-class model using the OneVsRestClassifier and gradient boosting (GradientBoostingClassifier) algorithms. The quality of the trained model was tested on test data. Statistical data processing, development of classification models, their testing and assessment of the quality of training were performed in the Jupyter Notebook environment v.6.5.2.Results. The training quality indicators of single-class models for recognizing BC subtypes were as follows: sensitivity in determining luminal A subtype (LA) was 67.0 %, luminal B subtype (LB) – 72.7 %, luminal B HER2-positive subtype (LBH) – 81.8 %, non-luminal HER2-positive (HER) and triple negative breast cancer (TNC) – 100 %. The specificity was 90.2 % for LA, 83.0 % for LB, 89.7 % for LBH, 98.3 % and 93.5 % in the cases of HER and TNC, respectively.The area under the ROC curve (AUC) depending on the molecular subtype was determined as follows: for LA – 0.88, for LB – 0.86, for LBH – 0.87, for HER – 0.96, and for TNC – 1.000. The multiclass model also showed low sensitivity values, except for the TNC (100 %) and HER (85.7 %) subtypes, low levels of positive predictive value for all subtypes, except for TNC (91.7 %), and high specificity and negative predictive value for all subtypes. The area under the ROC curve for the multiclass model was for the subtypes: LA – 0.88, LB – 0.86, LBH – 0.86, HER – 0.95 and for TNC – 1.00.Conclusion. The possibility of using certain combinations of diagnostic features obtained as a result of radiation diagnostic methods to determine the probability of a molecular biological subtype of BC was proven. This indicates the presence of prerequisites for the creation of a new diagnostic tool for typing BC using classification models of artificial intelligence. In the future, its implementation will reduce the likelihood of an error in determining the molecular biological subtype of BC, especially in situations where the doctor»s opinion and the results of the immunohistochemical study do not coincide.

Simple SummaryTriple-negative breast cancer (TNBC) does not express estrogen or progesterone hormone receptors and does not overexpress the HER2 transmembrane receptor. Treatment of TNBC is challenging due to the lack of these targets for specific therapies. The identification of new targets requires a better understanding of molecular mechanisms underlying the development of TNBC. Here, we show that the embryonic isoform of human RNA polymerase (Pol) III plays an important role in the development and spread of TNBC, as well as in the expression of tumor-specific proteins. Deletion of POLR3G, the specifying subunit of the embryonic isoform of Pol III in a TNBC cell line resulted in reduced tumor growth, suppressed metastasis, and induced expression of transcription factors not present in TNBC but characteristic of other types of breast cancer. Targeting POLR3G expression or processes regulated by POLR3G expression may therefore be useful for the treatment of TNBC in the future.RNA polymerase (Pol) III transcribes short untranslated RNAs that contribute to the regulation of gene expression. Two isoforms of human Pol III have been described that differ by the presence of the POLR3G/RPC32α or POLR3GL/RPC32β subunits. POLR3G was found to be expressed in embryonic stem cells and at least a subset of transformed cells, whereas POLR3GL shows a ubiquitous expression pattern. Here, we demonstrate that POLR3G is specifically overexpressed in clinical samples of triple-negative breast cancer (TNBC) but not in other molecular subtypes of breast cancer. POLR3G KO in the MDA-MB231 TNBC cell line dramatically reduces anchorage-independent growth and invasive capabilities in vitro. In addition, the POLR3G KO impairs tumor growth and metastasis formation of orthotopic xenografts in mice. Moreover, KO of POLR3G induces expression of the pioneer transcription factor FOXA1 and androgen receptor. In contrast, the POLR3G KO neither alters proliferation nor the expression of epithelial–mesenchymal transition marker genes. These data demonstrate that POLR3G expression is required for TNBC tumor growth, invasiveness and dissemination and that its deletion affects triple-negative breast cancer-specific gene expression.

Breast cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-related death among women. Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. TNBC shows a high capacity for early metastasis and leads to worse clinical outcomes than other breast cancer subtypes due to the lack of specific therapeutic targets. We are looking to develop cancer-specific therapeutic targets for TNBC. Many types of cancer cells including TNBC cells rely on glutamine as carbon and nitrogen source to fuel unchecked growth. We screened for key genes in regulating glutamine metabolism in a panel of breast cancer cell lines. This screen identified the mRNA and protein levels of solute carrier family 38 member 3 (SLC38A3), a glutamine transporter, to be upregulated in human breast cancer cell lines, especially in TNBC cell lines. The TCGA breast cancer patient database also showed that SLC38A3 mRNA is overexpressed in invasive ductal breast carcinoma tissues, and it is even higher in TNBC relative to other breast cancer subtypes. To test the biological role of SLC38A3 protein in TNBC cells, we performed loss-of-function experiments in multiple TNBC cell lines. Silencing of SLC38A3 decreased cellular glutamate, glutamine and alanine levels. Silencing of SLC38A3 also activated apoptosis, and suppressed cell viability, migration and invasion in several TNBC cell lines. Interestingly, silencing of SLC38A3 increased the activity of glycogen synthase kinase 3-β (GSK3β) which promoted the degradation of β-catenin, leading to the decrease of the mRNA levels of epithelial-to-mesenchymal-transition (EMT)-associated transcription factors in TNBC cell lines. In summary, we showed that SLC38A3 is overexpressed in TNBC and promotes breast cancer migration and invasion via GSK3β/β-catenin/EMT pathway, which could be a novel therapeutic target for breast cancer. Citation Format: Zheqiong Tan, Caitlin M. Tressler, Kanchan Sonkar, Kristine Glunde. Glutamine transporter SLC38A3 promotes breast cancer migration via GSK3beta/beta-catenin/EMT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB207.

Background: Triple negative breast cancer (TNBC) has a relative paucity of effective targeted therapies compared to other molecular subtypes, and with poor prognosis. Thus, new targeted therapeutics are needed. ONC201 is a first-in-class small molecule that induces caspase-mediated apoptosis in cancers. It recently showed efficacy in pediatric glioma, harboring the potential to be translated in advanced breast cancer. Here we evaluated the efficacy of ONC201 in TNBC cell lines, investigated proteomic/genomic markers that predict efficacy, and performed a synthetic lethal RNAi screening to identify rational combinational targets. Method: The CellTiter-Blue (CTB) cell viability or sulforhodamine B assay was used to measure the range of half-maximal inhibitory concentrations (IC50) of ONC201 in TNBC cell lines. 2D/3D high-throughput RNAi kinome screening was performed using ONC201-sensitive CAL51, resistant HCC70 cells. The top genes noted to augment ONC201 sensitivity by siRNA were analyzed using both protein-protein interactome analysis (STRING V.11) and Ingenuity Pathway Analysis (IPA) to identify contributing canonical pathways. Combinational anti-proliferative activity of ONC201 and target inhibitors were evaluated by CTB cell viability assay and quantified by CalcuSyn software. Identified potential partners were subsequently tested using 3D ex vivo spheroid assay, tumor xenograft animal model to measure tumor growth inhibition (TGI). Five NOD/SCID mice were treated per each group. Mice were treated with 50mg/kg twice daily dosing of ONC201, and/or 25mg /kg of palbociclib after tumor grew to 100mm3. Reverse phase protein array (RPPA) of both ONC201 treated and non-treated TNBC cell lysates was performed to identify pathways affecting the sensitivity to ONC201 by comparing the effect of protein level changes (individual and set) using 3-way ANOVA, R program. Results: Twenty TNBC cell lines exhibited varying IC50 values (2 μM to 40 μM). No correlation between Vanderbilt subtypes and IC50 was observed. RNAi kinome screening identified 65 overlapping target kinases. Pathway analyses revealed PI3K/Akt, MEK/ERK, CDK 2/4/6 as potential combination therapeutic partners. In the in vitro study, the MEK inhibitor trametinib and CDK 4/6 inhibitor palbociclib consistently showed synergistic TGI with ONC201 (combination index values [CI] = 0.1 - 0.7). AKT inhibitor MK-2206 showed moderate combinational TGI (CI = 0.5 - 0.9). PI3K inhibitors PF04691052, buparlisib, and dactolisib showed cell type-specific combinational TGI. RPPA analysis did not reveal direct apoptosis-regulators mediators of sensitivity to ONC201, yet identified 7 potential predictive markers (fibronectin, pHER2, PAR, PLK1, pRb, SOD2, and EMA) of resistance/sensitivity. It also provided the rationale to study CDK 4/6 inhibition as a combination partner. Ex vivo study of ONC201/palbociclib (CDK 4/6 inhibitor) treatment inhibited of the colony formation in CAL51 cells. Interestingly, ONC201/palbociclib did not induce synergy in HCC70 cells, while the ONC-201/trametinib pair was synergistic in both cell lines. CAL51 tumor xenograft in vivo study confirmed the synergy of ONC201/palbociclib. Conclusion: MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib demonstrated synergy with ONC201 yet palbociclib only in ONC201 sensitive CAL51. Detailed mechanisms of synergy are being investigated that can further guide clinical translation. Citation Format: Bora Lim, Christine Peterson, Elin Cho, Alexander Davis, Troy Pearson, Huey Liu, Minha Hwang, Debu Tripathy, Naoto T Ueno, Jangsoon Lee. ONC201 synergistically induces cell death in triple negative breast cancer with CDK 4/6 and MEK inhibition [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-14.