Future perspective of clinical usefulness of metabolic and mitochondrial heterogeneity in triple-negative breast cancers.

The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6) in the metabolic adaptation of rapidly growing versus invasive TNBC cells as well as in Lapatinib resistant (Lap-R) TNBC cells. AnxA6 expression in cultured TNBC cell lines, Lap-R cells and in crude isolates of mitochondria was determined by Western blotting and/or immunofluorescence assays. The oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and mitochondria activity were assessed using the Seahorse XF Analyzer. Intracellular metabolites were detected by 1H-NMR analysis. Using model proliferative basal-like and invasive mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. Down regulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent cellular energy phenotype. We further show that AnxA6-depletion was associated with decreased lipid droplet accumulation and the lipolytic phenotype by enhancing the uptake and mitochondrial fatty acid oxidation for ATP production. Chronic Lapatinib-induced up regulation of AnxA6 in AnxA6-low TNBC cells reversed their lipolytic to a more lipogenic/glycolytic metabolic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies significant metabolic adaptation during stress and/or chronic Lapatinib treatment and provide additional insights into the potential of AnxA6 as a biomarker for not only therapeutic intervention but also the metabolic subtyping of TNBC subsets. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB127.

BACKGROUND: Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in only 45% of patients. TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. Therefore, understanding the mechanisms of resistance to standard chemotherapy is essential. Our previous studies found mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual tumor cells that survived after NACT. Mitochondria are morphologically plastic and dynamic organelles that continuously cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. Mitochondrial fusion is thought to support OXPHOS through maintenance of the mitochondrial genome (mtDNA) which encodes 13 subunits of electron transport chain complexes. On the other hand, mitochondrial fission can be the precursor to mitophagy, the selective degradation of damaged mitochondria. We are investigating how mitochondrial structure dynamics impact metabolic states driving chemoresistance in TNBC. Methods We used a previously characterized orthotopic patient-derived xenograft (PDX) mouse model derived from the primary tumor of a treatment-naïve metastatic TNBC patient. After treatment with a standard NACT regimen, combined Adriamycin (aka doxorubicin) and cyclophosphamide (AC), we observed initial partial response followed by tumor regrowth. We collected PDX tumors at three time points: pre-treatment, post-AC residual (when tumors reached the volume nadir), and post-AC tumors that later regrew to the starting tumor volume. We used transmission electron microscopy (TEM) and immunohistochemical (IHC) analysis of human mitochondria to assess mitochondrial morphology and mass, respectively. For in vitro assays, we assessed mitochondria morphology, mtDNA content, and OXPHOS activity in TNBC cells treated with two different types of chemotherapeutic drugs, DNA-damaging agents (doxorubicin and carboplatin) and a microtubule-stabilizing agent (paclitaxel). To examine metabolic adaptations upon chemotherapy, we conducted 13C-glucose and glutamine flux metabolomics. To pharmaceutically perturb mitochondrial morphology, we used Mdivi-1 and Silibinin, known to induce mitochondrial fusion and fission, respectively. We assessed cell growth by Incucyte real-time imaging. Results: Analyses of residual PDX tumors after in vivo AC treatment revealed increased mitochondrial content, and increased average mitochondrial length in residual tumor cells. Metabolomic analysis of TNBC cells revealed all three chemotherapies induced glucose, but not glutamine, flux through the TCA cycle. Flux through glycolysis was not affected by chemotherapies. Treatment with doxorubicin and carboplatin, increased mitochondrial elongation, mtDNA content, and OXPHOS activity. Conversely, paclitaxel treatment reduced mitochondrial length and OXPHOS activity. We observed Mdivi-1 induced fusion concomitant with increased OXPHOS activity, while treatment with Silibinin reduced fusion and decreased OXPHOS. We next tested if induction of mitochondrial fission enhanced chemosensitivity. Sequential treatment of TNBC cells with DNA-damaging chemotherapeutics followed by Silibinin perturbed OXPHOS and resulted in enhanced cell killing. Conclusion: These findings establish a functional role for mitochondrial structure in chemotherapeutic response and metabolic reprogramming, which may confer a survival advantage to TNBC cells. Given the increased chemosensitivity we observed after pharmacologic inhibition of mitochondrial fusion, our study suggests perturbing mitochondrial structure adaptations may provide an opportunity to overcome TNBC chemoresistance in the neoadjuvant setting. Citation Format: Lily Mokryun Baek, Junegoo Lee, James P. Barrish, Bora Lim, Jeffrey T. Chang, Phillip L. Lorenzi, Weston Porter, Gloria V. Echeverria. Morphological and functional plasticity of mitochondria in chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-08.

Background TNBC comprises 10-20% of breast cancers. Lehmann et al. identified 6 TNBC subtypes by gene expression profiling: BL1, BL2, IM, M, MSL & LAR. Despite the diversity of TNBC, standard of care is combination chemotherapy as in non TNBCs. Identification of chemosensitive TNBC subtypes is necessary. Chemoresistant TNBCs need alternative targets to improve treatment strategies. Aims 1. To validate an IHC biomarker panel to define molecular subtypes of TNBC 2. To correlate molecular subtypes with prognosis to identify appropriate therapy 3. To improve diagnostic tools to individualize therapy based on TNBC subtypes Methods A TMA was constructed of 197 TNBCs diagnosed from 1999 - 2014. An 8-protein IHC panel was developed to identify TNBC subtypes on FFPE tissue. The panel includes markers for key pathways to discriminate between 6 subtypes: AR, Bcl2, c-myc, TIE1, PDGFC, MMP2, Il2R, MSH2. To date, AR & Bcl2 have been stained and assessed, together with p53 & Ki67. 10% was used as the cut off for positivity. Clinical data were obtained from hospital records and incorporated in the database. Results On initial observation, AR+ tumors had an older age at diagnosis than AR- (60 v 56), lower rates of family history (36 v 87%) and longer DFS (31 v 21 months). Bcl2+ tumors had a younger age at diagnosis than Bcl2- (55 v 59), lower recurrence rates (25 v 31%) and longer DFS (33 v 15 months). High Ki67 tumors had a younger age at diagnosis than low Ki67 (56 v 65), higher rates of family history (30 v 14%), lower recurrence rates (16 v 25%) and longer DFS (30 v 2 months). By the time of presentation, the entire panel of 8 proteins will be analyzed. The clinicopathological association of specific TNBC subtypes and the impact of TNBC subtype on chemotherapy response will be statistically assessed. Analysis will include response, duration, DFS and OS. This study will ultimately correlate TNBC molecular subtypes with prognosis to aid clinical decision making, individualize therapies and improve patient outcomes. n = 197AR +AR -Bcl2 +Bcl2 -Ki67 >10%Ki67 <10%P53 +P53 -n141429963160149164%7%72%50%32%30%7%46%32%Median Age6056555956655755Range37-7729-9029-8431-9229-8235-9029-9030-88Family Historyn51232916182313%36%87%29%25%30%14%3%20%BRCA Mutation06331134BRCA 1-3121122BRCA 2-321--12NACT010544364PR-8324144SD-111-11-POD-111-11-Recurrencen3 (of 12)38 (of 138)25 (of 98)18 (of 58)9 (of 58)3 (of 12)22 (of 88)18 (of 62)%25%28%25%31%16%25%25%29%Median DFS312133153022731Range30-342-549-542-384-542-22-549-52Median OS3537373628363438Range5-890-1342-1341-1331-1379-1261-1340-137 Citation Format: Elaine M. Walsh, Aliaa Shalaby, Laura Murillo, Mark Webber, Michael Kerin, Sharon Glynn, Grace Callaghy, Helen Ingoldsby, Maccon Keane. Identification of triple negative breast cancer (TNBC) subtypes by an immunohistochemistry (IHC) panel with impact on clinical outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1708. doi:10.1158/1538-7445.AM2015-1708

Breast cancer (BC) is the most frequent female cancer and a leading cause of female deaths worldwide. BC-related mortality rates are high among African American (AA) women despite the low incidence rates of breast cancer observed in this population compared with Caucasian Americans (CA). The triple negative breast cancer (TNBC) subtype lacks expression of three biomarkers used to clinically classify BC, and thus TNBCs cannot be treated with traditional receptor therapies. Moreover, as TNBC is biologically aggressive and women diagnosed with TNBC have poor outcomes. Interestingly, TNBC is most prevalent in young women of African Ancestry (WAA) compared to women of other ethnicities, but the cause of this racial disparity remains unknown. Recent studies in our lab revealed that the transcription factor Kaiso is highly expressed in TNBC tissues of WAA patients compared with those from Caucasian patients, suggesting a role for Kaiso in TNBC racial disparity. Intriguingly, our lab and others have also reported a correlation between high Kaiso expression, poor overall survival of AA BC patients compared with Caucasian patients, and increased TNBC aggressiveness/metastasis that is in part mediated via the TGFβ signaling pathway. Notably, Kaiso has also been implicated in tumor cell migration via its regulation of the tumor-suppressing microRNA-31 (miR-31) in prostate cancer cells. Remarkably, the pleiotropic miR-31 functions to suppress metastasis and its expression has been shown to be inversely correlated with aggressive breast tumor metastasis. Although Kaiso has been implicated in epithelial-to-mesenchymal transition (EMT) and TNBC metastasis, Kaiso's exact roles in the regulation of miRNAs in the context of TNBC remains to be elucidated. Using chromatin immunoprecipitation (CHIP) analysis, we found that Kaiso binds to the miR-31 and miR-200 promoters, and we detected increased expression of these microRNAs in Kaiso-depleted TNBC cells using qRT-PCR analysis. Furthermore, using immunoblot analysis, we found that Kaiso depletion resulted in reduced expression of the actin remodelling protein WAVE3, which is a downstream target of both miR-31 and miR-200. Consistent with these molecular changes, transfection of TNBC cells with miR-31 and miR-200 mimics resulted in reduced migration of these cells compared to control TNBC cells as assessed via migration assays. These data suggest that Kaiso regulates miR-31 and miR-200 in TNBC cells, and promotes TNBC cell migration via downregulation of these miRNAs. Ongoing studies seek to assess and correlate miR-31 and miR-200 expression with Kaiso expression in TNBC tissues of WAA. Together, our findings raise the exciting possibility that Kaiso may be developed as a potential target for the treatment of TNBC patients. Citation Format: Rayner LGA, Bassey-Archibong BI, Jaber S, Daniel JM. Kaiso regulates miRNA-31 and miRNA-200 expression in triple negative breast cancer (TNBC) cells [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 P5-07-06.

Background: Triple negative breast cancers (TNBC) are more aggressive, have a worse prognosis, and few targeted therapies compared to other BC subtypes. TNBC is molecularly heterogeneous, with at least 4 distinct subtypes: basal-like immune-activated (BLIA), basal-like immunosuppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES). The molecular subtyping gene signature, BluePrint (BP), classifies breast tumors into Luminal, HER2, or Basal subtype based on the assessment of downstream signaling pathways and independently of IHC expression. Compared to IHC-defined TNBC, a higher frequency of BLIS or BLIA subtypes and fewer LAR or MES tumors were reported in BP-defined Basal tumors. To advance our understanding of TNBC heterogeneity, we evaluated the relationship between gene expression signatures, TNBC subtype and BluePrint, in IHC-defined TNBC. Methods: The FLEX registry (NCT03053193) is an ongoing, prospective study evaluating primary tumors from stage I-III BC patients who receive the risk of distant recurrence gene signature, MammaPrint (MP), and BP testing and consent to clinically annotated full transcriptome data collection. This analysis includes 204 IHC-defined TNBC patients. TNBC subtypes BLIA, BLIS, LAR, and MES were derived using an adjusted version of the Burstein centroid signature. BP classified patient samples into Luminal, HER2, and Basal subtypes. A proportion of tumors may exhibit a secondary but less pronounced activated pathway or BP subtype. Therefore, each BP subtype was divided into single activated or mixed subtype based on BP indices. Results: Of 204 TNBC tumors, 84% were classified as Basal by BP, most of which were BLIS (65%), followed by BLIA (22%), with a low frequency of MES (8%) and LAR (5%) subtypes (Table). Approximately 14% of TNBCs were reclassified as Luminal by BP, most of which were LAR (76%), whereas 24% were MES. Clustering analysis revealed similar gene expression profiles between Basal-BLIS and Basal-BLIA tumors. Interestingly, the transcriptional profile of Basal-MES and Basal-LAR tumors were similar to Luminal-MES and Luminal-LAR tumors. BP Basal indices distinguished between different TNBC subtypes. The Basal pathway was predominantly activated in 90% of BP Basals (single activated tumors), most of which were either BLIS or BLIA (96%), whereas 10% of BP Basals were mixed subtype and more likely to classify as LAR (53%) or MES (35%). Approximately 25% of the BP Basal gene signature overlapped with the TNBC subtype gene signature. Expression of 18 and 12 genes out of 28 genes that make up the BP basal signature were significantly different in Basal-BLIA/BLIS compared to LAR or MES, respectively (P < 0.05). PRR15 and CAPN13 were significantly differentially expressed between LAR and MES within Basals. Conclusion: BP reclassified a subgroup of TNBC tumors to Luminal, explaining the discrepancy in the distribution of TNBC subtypes between IHC-defined TNBC and BP Basal tumors. Furthermore, BP indices distinguished between single activated and mixed subtypes, which correlated with different TNBC subtypes. These data suggest that molecular classification by BP adds further precision in classifying TNBC patients and sheds new light on the heterogeneity of these tumors. These findings have clinical implications in stratifying patients and identifying successful targeted treatment options. Future studies are warranted to investigate treatment response and prognosis in these molecular subgroups. BasalLuminalHER2TotalBLIA370037BLIS11100111LAR1022335MES147021Total172 (84.3%)29 (14.2%)3 (1.5%)204 (100%) Citation Format: Virginia G. Kaklamani, Cathy Graham, Karen L. Tedesco, Abirami Sivapiragasam, Jennifer A. Crozier, Apurva N. Shah, Yuan Yuan, Josien Haan, Andrea Menicucci, Michelle L. Bolner, Shiyu Wang, Lorenza Mittempergher, Erin Yoder, William Audeh, FLEX Investigators' Group. Using blueprint to elucidate the molecular heterogeneity of triple negative breast cancers [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 PS18-05.

BackgroundAmong breast cancers, the triple-negative breast cancer (TNBC) subtype has the worst prognosis with no approved targeted therapies and only standard chemotherapy as the backbone of systemic therapy. Unique metabolic changes in cancer progression provide innovative therapeutic opportunities. The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), and MET receptor are highly expressed in TNBC, making both promising therapeutic targets. RTK signaling profoundly alters cellular metabolism by increasing glucose consumption and subsequently diverting glucose carbon sources into metabolic pathways necessary to support the tumorigenesis. Therefore, detailed metabolic profiles of TNBC subtypes and their response to tyrosine kinase inhibitors may identify therapeutic sensitivities.MethodsWe quantified the metabolic profiles of TNBC cell lines representing multiple TNBC subtypes using gas chromatography mass spectrometry. In addition, we subjected MDA-MB-231, MDA-MB-468, Hs578T, and HCC70 cell lines to metabolic flux analysis of basal and maximal glycolytic and mitochondrial oxidative rates. Metabolic pool size and flux measurements were performed in the presence and absence of the MET inhibitor, INC280/capmatinib, and the EGFR inhibitor, erlotinib. Further, the sensitivities of these cells to modulators of core metabolic pathways were determined. In addition, we annotated a rate-limiting metabolic enzymes library and performed a siRNA screen in combination with MET or EGFR inhibitors to validate synergistic effects.ResultsTNBC cell line models displayed significant metabolic heterogeneity with respect to basal and maximal metabolic rates and responses to RTK and metabolic pathway inhibitors. Comprehensive systems biology analysis of metabolic perturbations, combined siRNA and tyrosine kinase inhibitor screens identified a core set of TCA cycle and fatty acid pathways whose perturbation sensitizes TNBC cells to small molecule targeting of receptor tyrosine kinases.ConclusionsSimilar to the genomic heterogeneity observed in TNBC, our results reveal metabolic heterogeneity among TNBC subtypes and demonstrate that understanding metabolic profiles and drug responses may prove valuable in targeting TNBC subtypes and identifying therapeutic susceptibilities in TNBC patients. Perturbation of metabolic pathways sensitizes TNBC to inhibition of receptor tyrosine kinases. Such metabolic vulnerabilities offer promise for effective therapeutic targeting for TNBC patients.

Breast cancers (BCs) remain the leading cause of cancer-related deaths among women worldwide. Among the different types of BCs, treating the highly aggressive, invasive, and metastatic triple-negative BCs (TNBCs) that do not respond to hormonal/human epidermal growth factor receptor 2 (HER2) targeted interventions since they lack ER/PR/HER2 receptors remains challenging. While almost all BCs depend on glucose metabolism for their proliferation and survival, studies indicate that TNBCs are highly dependent on glucose metabolism compared to non-TNBC malignancies. Hence, limiting/inhibiting glucose metabolism in TNBCs should curb cell proliferation and tumor growth. Previous reports, including ours, have shown the efficacy of metformin, the most widely prescribed antidiabetic drug, in reducing cell proliferation and growth in MDA-MB-231 and MDA-MB-468 TNBC cells. In the current study, we investigated and compared the anticancer effects of either metformin (2 mM) in glucose-starved or 2-deoxyglucose (10 mM; glycolytic inhibitor; 2DG) exposed MDA-MB-231 and MDA-MB-468 TNBC cells. Assays for cell proliferation, rate of glycolysis, cell viability, and cell-cycle analysis were performed. The status of proteins of the mTOR pathway was assessed by Western blot analysis. Metformin treatment in glucose-starved and 2DG (10 mM) exposed TNBC cells inhibited the mTOR pathway compared to non-treated glucose-starved cells or 2DG/metformin alone treated controls. Cell proliferation is also significantly reduced under these combination treatment conditions. The results indicate that combining a glycolytic inhibitor and metformin could prove an efficient therapeutic approach for treating TNBCs, albeit the efficacy of the combination treatment may depend on metabolic heterogeneity across various subtypes of TNBCs.

561 Background: Limited cell line and human data suggest that TNBCs characterized as mesenchymal and luminal androgen receptor (LAR) commonly have alterations in the PI3K pathway. More data is needed to better characterize the role of the PI3K pathway across TNBC subtypes. Methods: Pre-treatment tumor biopsies were collected from operable TNBC patients (pts) enrolled on a clinical trial of genomically tailored NAT (ARTEMIS; NCT02276443). Tumors were categorized into 5 groups using the Pietenpol criteria: basal-like (BL) comprised of BL-1 and BL-2, mesenchymal and mesenchymal stem-like (M), immunomodulatory (IM), LAR, or unspecified (UNS). Using whole exome sequencing data, variants (single nucleotide polymorphisms and insertions/deletions) and copy number variations (CNVs) were identified in 32 genes known to activate the PI3K pathway. Results: Tumor subtyping and pathologic response to NAT was available in 127 pts (clinical stage I: 9; II: 84; III: 34). PI3K pathway alteration defined as a variant in one of the evaluated genes and/or deletion of PTEN was seen in 76 (60%) tumors. The most frequent alterations were: PTEN deletion (21%), PIK3CA variant (11%), and PIK3R1 variant (8%). PI3K alteration and residual cancer burden (RCB) rates across TNBC subtypes are shown in the table. There was a significant difference in pathologic complete response (pCR)/RCB 0 rate after NAT across TNBC subtypes (chi2 test; P = 0.02). There was a significant difference in the incidence of PI3K pathway alteration across TNBC subtypes (chi2 test; P < 0.01). Overall, the presence of PI3K alteration was not associated with pCR (Fisher exact test; P = 0.85). Pts with M tumors had a higher rate of substantial residual disease (RCB II-III) after NAT. Presence of PI3K pathway alteration was common in the M subtype and associated with RCB II-III (82% in PI3K-altered vs 33% in wild-type tumors; Fisher exact test; P = 0.02). Presence of PI3K pathway alteration was common but not associated with response in the LAR subtype. Conclusions: The incidence of PI3K pathway alteration varied by TNBC subtype but was not associated with pathologic response to NAT with the exception of increased substantial residual disease (RCB II-III) in the M subtype. [Table: see text]

Background: Patients with triple negative breast cancer(TNBC) are known to have poor prognosis and derive no benefit from endocrine therapy or targeted treatments. Using a database from a multicenter registry in Korea, we present the clinical features and prognosis for TNBC with other subtypes of breast cancer and clinicopathologic variables that influence the 3-year survival of the TNBC patients. Materials and Methods: From 1993 to 2008, patients diagnosed with breast cancer who were registered to the Korean Breast Cancer Society Registry were analyzed retrospectively. A cohort of 26,767 patients were divided in four groups: luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), HER2 overexpression (ER-, PR-, HER2+), and triple negative (ER-, PR-, HER2-). Clinicopathologic features such as age, tumor size, nodal status, p53, ki-67 expression and survival were evaluated. Results: The luminal A (14437 patients, 53.9%) subtype was the largest in our study sample, as compared with luminal B (3517 patients, 13.1%), ER-/HER2+ (3227 patients, 12%), and TNBC (5586 patients, 21%) subtypes. Compared with luminal A subtype, TNBC correlated with younger age and more aggressive characteristics, such as larger size, more lymph node metastasis, and higher proliferation rate. Moreover, TNBC correlated with poor overall survival and breast cancer-specific survival. The hazard rate showed a peak at 24 months for the TNBC subtype, but after 60 months, the risk was similar to that of the luminal A subtype. Higher T, N stage and histologic grade, and lymphatic and vascular invasion showed poor prognosis in TNBC patients, but on multivariate analysis only histologic grade and ki-67 status were related to poor prognosis. Young age was related to poor prognosis in the luminal A subtype, however, age was not related to prognosis in the TNBC subtype. Of the 5586 TNBC patients, 282 patients (7.11%) expired within 3 years of diagnosis. T and N stage, and grade was significantly associated with prognosis on multivariate analysis. Discussion: TNBC subtype is characterized by a younger age with poorer outcome. However, younger age is not related to prognosis, and mortality risk decreases to that of the luminal A subtype, which is known to have the best prognosis after a few years. The underlying biology of the TNBC subtype is important, and further studies to discover novel biomarkers to predict prognosis and target treatments for the TNBC subtype are necessary. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-05-01.

Simple SummaryExisting chemotherapy treatments for breast cancer patients are high on toxicity. There are very limited options available for triple-positive breast cancer (TPBC) patients, and there have not been any major breakthrough for targeted therapy for triple-negative breast cancer (TNBC) patients. Therefore, there is a need to identify common therapeutic targets for breast cancer patients. In this manuscript, we compared the sphingolipid profiles of cancer cell lines representing TPBC and TNBC, and correlated these profiles with the proliferation and migration properties the of cell types. We then associated the sphingolipid profiles for each subtype specific cell line with transcriptional and translational expression of corresponding metabolizing enzymes. Our results suggested that ceramide kinase (CERK) that catalyzes the synthesis of ceramide-1-phosphates from ceramides is dysregulated in both cell types. We also showed that the targeting of CERK at transcriptional level by siRNA therapeutics or inhibiting the CERK activity by hydrogel-mediated delivery of chemical inhibitors can be an effective strategy to slow down the tumor progression. Therefore, CERK emerges as a potential therapeutic target that can be explored further for cancer therapy.Sphingolipids are key signaling biomolecules that play a distinct role in cell proliferation, migration, invasion, drug resistance, metastasis, and apoptosis. Triple-negative (ER−PR−HER2−) and triple-positive (ER+PR+HER2+) breast cancer (called TNBC and TPBC, respectively) subtypes reveal distinct phenotypic characteristics and responses to therapy. Here, we present the sphingolipid profiles of BT-474 and MDA-MB-231 breast cancer cell lines representing the TPBC and TNBC subtypes. We correlated the level of different classes of sphingolipids and the expression of their corresponding metabolizing enzymes with the cell proliferation and cell migration properties of BT-474 and MDA-MB-231 cells. Our results showed that each cell type exhibits a unique sphingolipid profile, and common enzymes such as ceramide kinase (CERK, responsible for the synthesis of ceramide-1-phosphates) are deregulated in these cell types. We showed that siRNA/small molecule-mediated inhibition of CERK can alleviate cell proliferation in BT-474 and MDA-MB-231 cells, and cell migration in MDA-MB-231 cells. We further demonstrated that nanoparticle-mediated delivery of CERK siRNA and hydrogel-mediated sustained delivery of CERK inhibitor to the tumor site can inhibit tumor progression in BT-474 and MDA-MB-231 tumor models. In summary, distinct sphingolipid profiles of TPBC and TNBC representing cell lines provide potential therapeutic targets such as CERK, and nanoparticle/hydrogel mediated pharmacological manipulations of such targets can be explored for future cancer therapeutics.

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

Breast cancer is a major cause of cancer related death. TRAIL has been of interest as a cancer therapeutic, but resistance has been observed in breast cancer and only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL. Small molecule ONC201 functions through the ATF4/CHOP pathway to upregulate TRAIL receptor DR5 (Kline et. al., Sci. Sig., in press, 2015), and through dual inhibition of Akt/ERK signaling to upregulate TRAIL (Allen et. al Sci. Trans. Med., 2013). We recently reported that ONC201 depletes colorectal cancer stem cell (CSC) markers and prevents formation of colonospheres. ONC201 also prevents colorectal CSCs from initiating growth of xenografted tumors (Prabhu et. al Can. Res., 2015). ONC201 has completed its first-in-human clinical trial in advanced solid tumors (Stein et al., 2015 AACR-NCI-EORTC meeting) and is being tested in multiple phase I/II clinical trials (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). We investigated ONC201 efficacy in TNBC (TRAIL-sensitive), BRCA1-deficient, and non-TNBC (TRAIL-resistant) cells. We demonstrate IC50 values for ONC201 in the low μM range for TNBC or BRCA1-deficient (n = 6) and non-TNBC cells (n = 5), doses that are achievable based on human PK. Importantly, ONC201 induces apoptotic death in both TNBC and non-TNBC cells. ONC201 depletes Aldefluor+ putative breast CSCs in vitro whereas paclitaxel does not. ONC201 inhibits growth of TNBC, BRCA1-deficient and non-TNBC CSC-like mammospheres while paclitaxel chemotherapy does not. We show ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 TNBC xenograft model. We investigated effects of ONC201 on mechanisms of TRAIL resistance in breast cancer cells, including expression of inhibitor of apoptosis (IAP) family proteins. We found that ONC201 mediates a decrease in XIAP, c-IAP1, and c-IAP2 expression in the breast cancer cells used. We also examined levels of DR5 using flow cytometry, as a mechanism of TRAIL resistance in breast cancer cells involves receptor endocytosis from the cell surface (Zhang et al., Mol Cancer Res., 2008). Interestingly, we observed greater increases in cell surface DR5 in TNBC cells than non-TNBC cells after ONC201 treatment. Our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC, BRCA1-deficient, and non-TNBC subtypes. ONC201 is efficacious against breast cancer cells regardless of the TRAIL sensitivity of the cells, and this efficacy may be mediated through mechanisms involving downregulation of IAP proteins and upregulation of cell surface death receptors. Furthermore, ONC201 may target paclitaxel-resistant breast CSCs. Our findings suggest unique targeting of multiple non-TNBC, BRCA1-deficient and TNBC cells and develop a preclinical rationale for the use of ONC201 as a treatment for breast cancer. Citation Format: Marie D. Baumeister, Jessica Wagner, Varun V. Prabhu, Christina LB Kline, Bora Lim, Josh E. Allen, David T. Dicker, Wafik S. El-Deiry. ONC201 induces cell death in triple negative, BRCA1-deficient and non-triple negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3012.

Triple-negative breast cancers (TNBC) are a subset of breast cancer that is highly aggressive and has a poor prognosis. The TNBC subtype has no effective molecular targeted therapies. Gene expression studies have identified a subset of TNBC that is enriched with androgen receptors (AR) and androgen receptor signaling. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Interference with androgen signaling in TNBC with AR-inhibiting drugs have shown a reduction in epithelial-to-mesenchymal transition (EMT), which is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal-like stem cells. Approximately one-third of TNBC expressed AR, and evaluation of AR-positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. Previous studies have shown that AR inhibition or AR knockdown significantly reduces migration and invasion and EMT in different TNBC cell lines. γ-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer effects and have little or no effect on normal cell viability. Studies show that treatment with 0-7 µM γ-tocotrienol reduced AR levels, proliferation, migration and invasion in a dose-responsive manner in TNBC MDA-MB-231 and MDA MB-453 cell lines. Results also show that treatment with 5 µM (MDA-MB-231) and 7 µM (MDA-MB-453) γ-tocotrienol induced a reversal in EMT cell biomarkers, as well as reversal in EMT morphological and behavioral characteristics in these TNBC cell lines. Western blot analysis show that similar treatment with γ-tocotrienol significantly decreased DHT-induced N-cadherin (mesenchymal cell biomarker) and increased expression in cytokeratin 18 and E-cadherin (epithelial cell biomarkers) in these cells. AR signaling is known to play an important role in stimulating DHT-induced EMT by stimulating the STAT3/Snail signaling pathway in MDA-MB231. However, results in the present study shows that γ-tocotrienol treatment significantly inhibited activation of STAT3 and Snail proteins. Additional immunocytochemistry experiments showed that γ-tocotrienol treatment significantly inhibited DHT-induced cytoskeleton changes in TNBC MDA-MB-231 and MDA MB-453 cell lines. In conclusion, these results demonstrated that γ-tocotrienol treatment inhibits AR expression and DHT-dependent EMT and cytoskeleton changes in TNBC cells. These findings suggest that AR may be a potential therapeutic target for treating both LAR and non-LAR TNBC subtypes. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Nayef Aldabaan, Tasmin A. Sultana, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 967.

Background. Triple negative breast cancer (TNBC) is a challenging both in the choice of therapies and clinical outcomes. In the present study, we investigated the potential prospect of a Chinese medicine formation, ShenLingLan, shown to have benefit to patients with cancer and able to influence the biological behaviour of cancer cells, on breast cancer cells in particular the differential response from TNBC and non-TNBC cells and, on the discovery that TNBC cells were particularly sensitive to the medicine, we went on to determine the signalling and mechanism of action. Methods. A panel of three TNBC (MDA MB-231, BT20 and BT549) and a panel of three non-TNBC (MCF-7, ZR 75-1 and T47D) cells were used. A soluble extract from ShenLingLan, designated as SLDM, was utilised during this study. The proliferation, cellular migration and adhesiveness were tested using conventional and biophysical methods. Signalling profiling was conducted using a protein kinase array platform (Kinexustm). Metabolic profiling was conducted using the Seahorse platform. Expression of insulin receptor (IR) and insulin-like growth factor receptor (IGFR) gene transcripts (quantitative transcript analysis) and proteins (IHC) were conducted using a fresh breast cancer cohort and tissue array, respectively. Results. SLDM had little effects on the growth of breast cancer cells. However, it had profound inhibitory effects on the migration of both TNBC and non-TNBC cells in a concentration dependent manner. Interestingly, TNBC cells were 5-20 times more sensitive than the non-TNBC cells in their migration and cell adhesion responses to SLDM. The protein array platform further revealed that, of the wide range of protein kinases, IR and IGRR1 were the most affected in that SLDM resulted in 25-50% reduction in the phosphorylation of IR and IR substrate in TNBC cells. SLDM also caused a contrasting response in IGFR1 phosphorylation in TNBC and non-TNBC cells. Metabolically, TNBC and non-TNBC cells responded to SLDM in very different fashions. For example, in TNBC cells SLDM resulted in a significant decrease in glycolytic activities, in particular that driven by insulin (30.2±12.2 pmol/min in control, 47.7±10 pmol/min with insulin and 35.4±4.2 pmol/min with insulin/SLDM, p=0.01). There is evidence that the mitochondria oxygen consumption (OCR) was also affected by SLDM in TNBC cells (p=0.01). These changes induced by SLDM were in clear contrast to non-TNBC cells which did not respond with significant reduction. Both TNBC and non-TNBC breast cancer tissues have higher IR staining than normal mammary tissues (p<0.001). TNBC tumours also demonstrated significantly more positive IR staining than the non-TNBC tumours (p=0.04). Conclusion. ShenLingLan has a profound inhibitory effect on the migration and cell-matrix adhesion of TNBC cells, with marked effect on the metabolics of these cells. This effect connects with reduction of the IR and IGFR activation, mainly through the reduction in glycolysis. Together with the clinical implication of IR and IGFR in breast cancer, ShenLingLan has an important role in the treatment of breast cancer with an emphasis in TNBCs. Citation Format: Jiang WG, Owen S, Ruge F, Gao Y, Wang H, Wei C, Wu Y, Davies E. The impact of the Chinese medicine ShenLingLan on triple negative breast cancer, the metabolic and signalling pathways and clinical implications [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 P3-06-10.

Despite recent advances in breast cancer therapeutics, mortality of metastatic triple negative breast cancer (TNBC) subtype remains high; due to their lack of hormone receptors expression for targeted therapy. Aberrant activation of Wnt/β-catenin signaling has been associated with breast cancers; where 40% of total breast cancers have elevated β-catenin levels with increased Wnt activity. Recently, we identified DEAD-box RNA helicase DP103 as a novel prognostic biomarker and metastasis-driving oncogene; highly expressed in TNBC subtype. Interestingly, we found high DP103 expression to be positively correlated with high β-catenin expression in clinical specimens (n=400). This led us to hypothesize a possible role of DP103 in modulating the Wnt/β-catenin pathway in TNBCs. Depletion of DP103 in metastatic TNBC cells decreases Wnt/β-catenin activity and expression of downstream Wnt target genes, while overexpression of DP103 increases Wnt activity. Depletion of DP103 also decreases phosphorylation of LRP6 and several important Wnt modulators required for downstream Wnt activation. Moreover, induction of Wnt/β-catenin signaling in Wnt responsive TNBC cells also significantly increased DP103 expression, indicating a possible positive feedback loop. Both canonical and non-canonical Wnt signaling is known to independently promote stem cell growth in mammospheres. Herein, we will also provide evidence on the role of DP103 in promoting breast cancer stem cell-like properties. Collectively, our data show a novel regulatory role of DP103 in the Wnt/β-catenin signaling pathway and in promoting breast cancer stem cell-like behavior, presenting itself as a potential drug target in TNBC patients. Citation Format: Cai W, Cheong JK, Edison E, Banerjee A, Tan TZ, Gaboury L, Yousef EM, Thiery JP, Lobie PE, Virshup DM, Yap CT, Kumar AP. DEAD-box RNA helicase DP103 as a novel regulator of Wnt/β-catenin signaling pathway and promotes cancer stem cell-like behavior in triple negative 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 P4-08-03.