Breast Cancer Patients Research Articles

The acyltransferase transmembrane protein 68 regulates breast cancer cell proliferation by modulating triacylglycerol metabolism.

Cellular carcinogenesis is often marked by the accumulation of lipid droplets (LDs) due to reprogrammed lipid metabolism. LDs are dynamic organelles that primarily store intracellular triacylglycerol (TAG) and cholesteryl esters (CEs). Transmembrane protein 68 (TMEM68), a potential modifier of human breast cancer risk and outcomes, functions as a diacylglycerol acyltransferase, synthesizing TAG. However, the specific roles of TMEM68 in breast cancer cells remain unclear. Gene expression profiling interactive analysis and survival analysis were conducted. TMEM68 was overexpressed or knockdown in breast cancer cells to assess its impact on cell proliferation, migration and invasion. Targeted quantitative lipidomic analysis and quantitative polymerase chain reaction were used to profile lipid alterations and examine gene expression related to lipid metabolism following changes in TMEM68 levels. TMEM68 gene was upregulated in breast cancer patients and higher TMEM68 levels were associated with poorer survival outcomes. Overexpression of TMEM68 increased breast cancer cell proliferation and invasion, whereas knockdown had minimal or no impact on reducing proliferation and invasion. Altering TMEM68 levels resulted in corresponding changes in TAG levels and cytoplasmic LDs, with overexpression increasing both and knockdown decreasing them. Lipidomic analysis revealed that TMEM68 regulated TAG levels and altered diacylglycerol content in breast cancer cells. Additionally, TMEM68 influenced the metabolism of glycerophospholipids, CEs and acylcarnitines. TMEM68 also modified the expression of key genes encoding enzymes related to neutral lipid metabolism, including TAG and CEs. TMEM68 is highly expressed in breast cancer and negatively correlated with survival. Its overexpression promotes breast cancer cell proliferation while knockdown has varied effects depending on TMEM68 levels. TMEM68 regulates intracellular TAG and LDs contents along with alterations in glycerophospholipids. These findings suggest that TMEM68 may drive breast cancer cells proliferation by modulating TAG and LD content.

Baseline gut microbiome alpha diversity predicts chemotherapy-induced gastrointestinal symptoms in patients with breast cancer.

Chemotherapy frequently causes debilitating gastrointestinal symptoms, which are inadequately managed by current treatments. Recent research indicates the gut microbiome plays a role in the pathogenesis of these symptoms. The current study aimed to identify pre-chemotherapy microbiome markers that predict gastrointestinal symptom severity after breast cancer chemotherapy. Fecal samples, blood, and gastrointestinal symptom scores were collected from 59 breast cancer patients before, during, and after chemotherapy. Lower pre-chemotherapy microbiome alpha diversity and abundance of specific microbes (e.g., Faecalibacterium) predicted greater chemotherapy-induced gastrointestinal symptoms. Notably, tumor and diet characteristics were associated with lower pre-chemotherapy alpha diversity. Lower baseline alpha diversity also predicted higher chemotherapy-induced microbiome disruption, which was positively associated with diarrhea symptoms. The results indicate certain cancer patients have lower microbiome diversity before chemotherapy, which is predictive of greater chemotherapy-induced gastrointestinal symptoms and a less resilient microbiome. These patients may be strong candidates for pre-chemotherapy microbiome-directed preventative interventions (e.g., diet change).

Non-coding RNAs modulation in breast cancer radioresponse: mechanisms and therapeutic implications.

Breast cancer is the most frequent type of cancer in women, with significant incidence and fatality rates. Radiation therapy is an important therapeutic option for breast cancer patients. However, tumor cells' resistance to radiation can limit therapy efficacy, resulting in recurrence and death. Non-coding RNAs (ncRNAs) are aclass of small RNA molecules that do not translate into proteins but can affect the translation of target mRNA. Several investigations on breast cancer have demonstrated abnormal expression of ncRNAs in response to radiation. Non-coding RNAs are essential in controlling numerous processes such as DNA damage response, cancer stem cell pathways, cell cycle regulation, cell death, and inflammation. Dysregulation of ncRNAs after irradiation influences radiosensitivity or radioresistance of breast cancer cells. Understanding the molecular mechanisms underlying Radiation response can lead to innovative treatment ways to reduce breast cancer radioresistance and increase radiotherapy's efficacy. This review summarizes current research on ncRNA dysregulation following irradiation and analyzes ncRNAs' function and mechanism in modifying breast cancer cell radiosensitivity and radioresistance.

Germline Variant Spectrum in Southern Italian High-Risk Hereditary Breast Cancer Patients: Insights from Multi-Gene Panel Testing

Hereditary breast cancer accounts for 5–10% of all cases, with pathogenic variants in BRCA1/2 and other susceptibility genes playing a crucial role. This study elucidates the prevalence and spectrum of germline variants in 13 cancer predisposition genes among high—risk hereditary breast cancer patients from Southern Italy. We employed next-generation sequencing (NGS) to analyze 254 individuals selected through genetic counseling. Pathogenic or likely pathogenic variants were identified in 13% (34/254) of patients, with 54% of these variants occurring in non-BRCA1/2 genes. Notably, we observed a recurrent BRCA1 c.4964_4982del founder mutation, underscoring the importance of population-specific genetic screening. The spectrum of variants extended beyond BRCA1/2 to include PALB2, ATM, TP53, CHEK2, and RAD51C, highlighting the genetic heterogeneity of breast cancer susceptibility. Variants of uncertain significance were detected in 20% of patients, emphasizing the ongoing challenge of variant interpretation in the era of multi-gene panel testing. These findings not only enhance our understanding of the genetic landscape of breast cancer in Southern Italy but also provide a foundation for developing more targeted, population-specific approaches to genetic testing and counseling, ultimately contributing to the advancement of precision medicine in oncology.

Perspectives on Quality of Life in Women with Breast Cancer

Objectives: The objectives of the study are to assess changes in the quality of life amongst breast cancer patients undergoing treatment at a cancer institute in Colombia. Materials and Methods: Analytical observational prospective cohort study in patients over 18 years of age diagnosed with breast cancer. Health-related quality of life (HRQoL) was analysed using the EQ-5D-3L questionnaire and a Visual Analogue Scale measured at diagnosis and after a 6-month follow-up. Sociodemographic and clinical factors were analysed using a logistic regression model, with STATA 16 software. Results: A total of 103 patients met the included criteria and were included in the study, with a median age of 56 years. According to the Tumour, Node, Metastasis classification of cancer stage, the majority of participants (35.92%) were in stage 2 of cancer. Multivariate analysis revealed that changes in HRQoL were significantly associated with age (odds ratio [OR] = 1.06, P = 0.001), radiotherapy (OR = 3.56, P = 0.038) and moderate anxiety and depression (OR = 5.54, P = 0.007). Conclusion: While the overall quality of life in women with breast cancer showed a slight improvement over the 6 months, older patients and those receiving radiotherapy experienced a greater decline in health perception.

The effect of cancer and cancer treatment on attention control: evidence from anti-saccade performance.

Cancer and cancer treatment have been associated with cognitive changes in survivorship, with forgetfulness and distractibility reported years post-treatment. Deficits in attention control may explain these difficulties. We assessed breast cancer survivors using a primary measure of attention control, the saccade/antisaccade task, to assess the effects of diagnosis and treatment. Saccade performance was studied in a cohort of breast cancer patients at two time points, (1) after surgery before adjuvant treatment and (2) approximately 2years after enrollment, and compared to non-cancer controls. Saccade performance was assessed in a prosaccade task as well as in visually guided and unguided antisaccade tasks. We assessed the frequency of directional errors and saccadic reaction time. Survivors were more likely than controls to make directional errors in an unguided antisaccade task, with older survivors exhibiting the most significant difficulties following adjuvant treatment. Survivor and control performance were much more similar in a visually guided antisaccade task. These results indicate a main effect of cancer diagnosis on attention control, with greater deficits following treatment and in older survivors. Deficits in attention control may lead to greater difficulties in the initial learning of information, explaining reports of forgetfulness in survivors. These findings underscore the enduring impact of cancer and its treatment on attention control, particularly highlighting that older breast cancer survivors may experience more pronounced difficulties with inhibitory control in daily life. Antisaccade performance may provide a useful metric for quantifying this impact.

A New Breast Cancer Discovery Strategy: A Combined Outlier Rejection Technique and an Ensemble Classification Method

Annually, many people worldwide lose their lives due to breast cancer, making it one of the most prevalent cancers in the world. Since the disease is becoming more common, early detection of breast cancer is essential to avoiding serious complications and possibly death as well. This research provides a novel Breast Cancer Discovery (BCD) strategy to aid patients by providing prompt and sensitive detection of breast cancer. The two primary steps that form the BCD are the Breast Cancer Discovery Step (BCDS) and the Pre-processing Step (P2S). In the P2S, the needed data are filtered from any non-informative data using three primary operations: data normalization, feature selection, and outlier rejection. Only then does the diagnostic model in the BCDS for precise diagnosis begin to be trained. The primary contribution of this research is the novel outlier rejection technique known as the Combined Outlier Rejection Technique (CORT). CORT is divided into two primary phases: (i) the Quick Rejection Phase (QRP), which is a quick phase utilizing a statistical method, and (ii) the Accurate Rejection Phase (ARP), which is a precise phase using an optimization method. Outliers are rapidly eliminated during the QRP using the standard deviation, and the remaining outliers are thoroughly eliminated during ARP via Binary Harris Hawk Optimization (BHHO). The P2S in the BCD strategy indicates that data normalization is a pre-processing approach used to find numeric values in the datasets that fall into a predetermined range. Information Gain (IG) is then used to choose the optimal subset of features, and CORT is used to reject incorrect training data. Furthermore, based on the filtered data from the P2S, an Ensemble Classification Method (ECM) is utilized in the BCDS to identify breast cancer patients. This method consists of three classifiers: Naïve Bayes (NB), K-Nearest Neighbors (KNN), and Support Vector Machine (SVM). The Wisconsin Breast Cancer Database (WBCD) dataset, which contains digital images of fine-needle aspiration samples collected from patients’ breast masses, is used herein to compare the BCD strategy against several contemporary strategies. According to the outcomes of the experiment, the suggested method is very competitive. It achieves 0.987 accuracy, 0.013 error, 0.98 recall, 0.984 precision, and a run time of 3 s, outperforming all other methods from the literature.

Worse Clinical and Survival Outcomes in Breast Cancer Patients Living in Puerto Rico Compared to Hispanics, Non-Hispanic Blacks, and Non-Hispanic Whites from Florida.

Herein, we report the characterization of four cohorts of breast cancer patients including (1) non-Hispanic Whites in Florida, (2) non-Hispanic Blacks in Florida, (3) Hispanics in Florida, and (4) Hispanics in Puerto Rico. Data from female breast cancer patients were collected from cancer registry (n = 9361) and self-reported patient questionnaires (n = 4324). Several statistical tests were applied to identify significant group differences. Breast cancer patients from Puerto Rico were least frequently employed and had the lowest rates of college education among the groups. They also reported more live births and less breastfeeding. Both Hispanic groups reported a higher fraction experiencing menstruation at age 11 or younger (Floridian Hispanics [38%] and Puerto Ricans [36%]) compared to non-Hispanic Whites (20%) and non-Hispanic Blacks (22%). Non-Hispanic Black and Puerto Rican women were significantly older at breast cancer diagnosis than their non-Hispanic White and Floridian Hispanic counterparts. The Puerto Rican and non-Hispanic Black groups more frequently had pathology stage T2 or higher primary breast tumors at diagnosis (non-Hispanic Whites [29%], non-Hispanic Blacks [39%], Floridian Hispanics [33%], Puerto Ricans [46%]). The Puerto Rican (73%, 95% CI [66, 82]) and non-Hispanic Black (79%, 95% CI [75, 84]) groups demonstrate reduced 5-year survival compared to non-Hispanic Whites (89%, 95% CI [86, 92]) and Floridian Hispanics (89%, 95% CI [86, 90]). These findings demonstrate that Puerto Rican breast cancer patients suffer significant breast cancer health disparities relative to non-Hispanic Whites and Hispanics from Florida similar to the disparities observed for non-Hispanic Blacks. Future work must seek to better understand and address these disparities.

Abstract A003: Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A003.

Pericardial Tamponade Two Years after Central Venous Catheter Implantation in a Patient with Breast Cancer: A Case Report

We report a rare and diagnostically challenging case of pericardial tamponade in a breast cancer patient, treated two years earlier. Echocardiogram and an enhanced computed tomography (CT) scan revealed a large mass in the right pericardium with indistinct borders. A positron emission tomography-computed tomography (PET-CT) scan showed no uptake of fluorine-18 fluorodeoxyglucose (18F-GDP) by the mass. Cardiac magnetic resonance imaging (MRI) with late gadolinium enhancement confirmed no signal enhancement within the mass and a clear border, decisively ruling out malignancy. This case highlights the importance of late gadolinium enhancement in this diagnostically challenging case and explores the pericardial tamponade development mechanism.

Performance of an AI-powered visualization software platform for precision surgery in breast cancer patients.

Surgery remains the primary treatment modality in the management of early-stage invasive breast cancer. Artificial intelligence (AI)-powered visualization platforms offer the compelling potential to aid surgeons in evaluating the tumor's location and morphology within the breast and accordingly optimize their surgical approach. We sought to validate an AI platform that employs dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to render three-dimensional (3D) representations of the tumor and 5 additional chest tissues, offering clear visualizations as well as functionalities for quantifying tumor morphology, tumor-to-landmark structure distances, excision volumes, and approximate surgical margins. This retrospective study assessed the visualization platform's performance on 100 cases with ground-truth labels vetted by 2 breast-specialized radiologists. We assessed features including automatic AI-generated clinical metrics (e.g., tumor dimensions) as well as visualization tools including convex hulls at desired margins around the tumor to help visualize lumpectomy volume. The statistical performance of the platform's automated features was robust and within the range of inter-radiologist variability. These detailed 3D tumor and surrounding multi-tissue depictions offer both qualitative and quantitative comprehension of cancer topology and may aid in formulating an optimal surgical approach for breast cancer treatment. We further establish the framework for broader data integration into the platform to enhance precision cancer care.

Mediator kinase inhibitors suppress triple-negative breast cancer growth and extend tumor suppression by mTOR and AKT inhibitors

Triple-negative breast cancers (TNBC) are treated primarily by chemotherapy and lack clinically validated therapeutic targets. In particular, inhibitors of the PI3K/AKT/mTOR pathway, abnormally activated in many breast cancers, failed to achieve clinical efficacy in TNBC due to the development of adaptive drug resistance, which is largely driven by the transcriptomic plasticity of TNBC. Expression of CDK8/19 Mediator kinases that control transcriptional reprogramming correlates with relapse-free survival and treatment failure in breast cancer patients, including TNBC. We now investigated how CDK8/19 inhibitors affect the growth of TNBC tumors and their response to mTOR and AKT inhibitors. In contrast to the effects of most anticancer drugs, all the tested human TNBC models (including patient-derived xenografts) responded to CDK8/19 inhibitors in vivo even when they did not respond in vitro. Furthermore, CDK8/19 inhibition extended the host survival of established lung metastases in a murine TNBC model, where the primary tumors were not significantly affected. CDK8/19 inhibitors synergized with an mTORC1 inhibitor everolimus and a pan-AKT inhibitor capivasertib in vitro and strongly potentiated these drugs in long-term in vivo studies. Transcriptomic analysis of tumors that responded or became adapted to everolimus revealed that drug adaptation in vivo was associated with major transcriptional changes in both tumor and stromal cells. Combining everolimus with a CDK8/19 inhibitor counteracted many of these changes and induced combination-specific effects on the expression of multiple genes that affect tumor growth. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors as a new type of drugs for TNBC therapy.

Abstract B012: HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy

Abstract The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but TNBC patients have a higher chance for relapse despite receiving increased doses of radiation compared to patients with other breast cancer subtypes. This study investigated the differential pathways in radioresistant TNBC tumors that impact recurrence. We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration and invasion were assessed using the scratch-wound assay and transwell assay, respectively. We assessed in vivo metastasis by injecting the cells in the mammary fat pad, tail vein, or left ventricle. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin b3 was assessed by inhibiting transcription using actinomycin D. Moreover, we implemented the newly developed InduroRT-mediated circRNA-sequencing (IMRT-seq) to identify the circular RNAs processed by HNRNPL that are present in radioresistant cells. We observed enhanced lung and bone metastasis, based on bioluminescent imaging, in both the spontaneous and vascular metastasis models for the radioresistant cells compared to the parental cells. Genset enrichment analysis identified mRNA stability and metabolism as upregulated pathways in radioresistant cells with HNRNPL, a ribonucleoprotein involved in mRNA stability, as one of the top genes. By knocking down HNRNPL, we observed decreased migration capacity of the radioresistant cells. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin b3 as a possible target. More specifically, knocking down HNRNPL decreased the mRNA stability of integrin b3 as assessed by actinomycin d treatment. By expressing integrin b3 in the HNRNPL-depleted cells, we were able to rescue the migration capacity of these cells. We believe a subset of HNRNPL-mediated circular RNAs are responsible for integrin b3 mRNA stability by sequestering miRNAs that target integrin b3. With IMRT-seq we are exploring the potential circRNA-miRNA interactions present in radioresistant TNBC responsible for its metastatic phenotype. The radioresistant cells derived from TNBC cell lines have an enhanced metastatic potential than the treatment-naive cell lines and our data suggest that RNA metabolism is pivotal to this phenotype. The upregulation of HNRNPL upon radiation by Nrf2 increases integrin b3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radioresistant metastasis provides a therapeutic approach to minimize TNBC recurrence. Citation Format: Ayush Kumar, Kensei Kishimoto, Christi Wisniewski, Shivam Goel, Arthur Mercurio. HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B012.

Abstract B013: Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression

Abstract Breast cancer (BC) is the most diagnosed cancer and the second leading cancer-related cause of mortality among women in the US. Despite advances in treatment, BC remains a challenge due to its metastatic potential. The most aggressive BC subtype, triple negative (TN), often harbors mutations in the tumor suppressor gene, p53. One of the effects of mutant p53 involves the inhibition of the tumor and metastatic suppressive functions of TAp63, a p53 family member. Loss of TAp63 leads to the formation of metastatic mammary adenocarcinomas in mice, in part through the regulation of a group of nine long non-coding RNAs (lncRNAs), that we identified and named TAp63 regulated oncogenic lncRNAs (TROLLs). We previously characterized two TROLLs, TROLL-2 and TROLL-3, as markers of tumor progression in multiple cancer types via the activation of the AKT pathway. Currently we are investigating the role of another member, TROLL-9. To investigate the biological role of TROLL-9, we performed in vivo experiments using preclinical mouse models. Specifically, MCF-DCIS, MCF-CA1D, and MDA-MB-231 triple negative breast cancer (TNBC) cells constitutively expressing TROLL-9 were generated and utilized for two mouse models: orthotopic xenografts for primary tumor growth and spontaneous metastasis. The former was designed to identify the potential role of TROLL-9 in tumor formation, whereas the latter involved the resection of a primary tumor and monitoring for metastases via bioluminescence imaging. Notably, TROLL-9 overexpression led to increased formation of lung and livers metastases in all these models, suggesting a role for TROLL-9 in the metastatic cascade. In order to elucidate the mechanism of function of TROLL-9, two complementary approaches were used. Firstly, pulldown using MS2-TRAP followed by mass spectrometry (MS) was performed and led to the identification of putative protein interactors. In parallel, Laser Capture Microscopy (LCM) of the lung metastases followed by RNA-sequencing was used to assess for any gene expression changes due to TROLL-9 overexpression. Integration of the two datasets is currently being performed to unveil the network of genes and proteins perturbed by TROLL-9. Furthermore, to understand the relevance in BC patients, TROLL-9 expression levels were analyzed in the TCGA BC dataset. Interestingly, differences in TROLL-9 levels across BC subtypes were observed, thus supporting its putative role in TNBC. Overall, our data suggest a role for TROLL-9 in BC progression. Our findings provide evidence for further characterizing its mechanism of function as a potential therapeutic target for the treatment of invasive BC, particularly TNBC harboring TP53 mutations. Citation Format: Avani A Deshpande, Marco Napoli, John H Lockhart, Christina L Carr, Jaden R Baldwin, Elsa R Flores. Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B013.

Evidence that CRISPR-Cas9 Y537S-mutant expressing breast cancer cells activate Yes-associated protein 1 to driving the conversion of normal fibroblasts into cancer-associated fibroblasts.

Endocrine therapy (ET) has improved the clinical outcomes of Estrogen receptor alpha-positive (ERɑ +) breast cancer (BC) patients, even though resistance to ET remains a clinical issue. Mutations in the hormone-binding domain of ERɑ represent an acquired intrinsic mechanism of ET resistance. However, the latter also depends on the multiple functional interactions between BC cells and the tumor microenvironment (TME). Here, we investigated how the most common Y537S-ERɑ mutation may influence the behavior of fibroblasts, the most prominent component of the TME. We conducted coculture experiments with normal human foreskin fibroblasts BJ1-hTERT (NFs), cancer-associated fibroblasts (CAFs), isolated from human BC specimens, and Y537S CRISPR-expressing MCF-7 BC cells (MCF-7YS). Mass spectrometry (MS) and Metacore analyses were performed to investigate how the functional interactions between BC cells/fibroblasts may affect their proteomic profile. The impact of fibroblasts on BC tumor growth and metastatic potential was evaluated in nude mice. Mutant BC conditioned medium (CM) affected the morphology/proliferation/migration of both NFs and CAFs. 198 deregulated proteins signed the proteomic similarity profile of NFs exposed to the YS-CM and CAFs. Among the upregulated proteins, Yes-associated protein 1 (YAP1) was the main central hub in the direct interaction network. Increased YAP1 protein expression and activity were confirmed in NFs treated with MCF-7YS-CM. However, YAP1 activation appears to crosstalk with the insulin growth factor-1 receptor (IGF-1R). Higher amount of IGF-1 were noticed in the MCF-7YS-CM cells compared to the MCF-7P, and IGF-1 immunodepletion reversed the enhanced YAP1 expression and activity. Mutant cells upon exposure to the NF- and CAF-CM exhibited an enhanced proliferation/growth/migration/invasion compared to the MCF-7P. MCF-7YS cells when implanted with CAFs showed an early relative increased tumor volume compared to YS alone. No changes were observed when MCF-7P cells were co-implanted with CAFs. Compared with that in MCF-7P cells, the metastatic burden of MCF-7YS cells was intrinsically greater, and this effect was augmented upon treatment with NF-CM and further increased with CAF-CM. YS mutant BC cells induced the conversion of fibroblasts into CAFs, via YAP, which represent a potential therapeutic target which interrupt the functional interactions between mutant cells/TME and to be implemented in the novel therapeutic strategy of a subset of metastatic BC patients carrying the frequent Y537S mutations.

The BrEasT cancer afTER-CARE (BETTER-CARE) programme to improve breast cancer follow-up: design and feasibility study results of a cluster-randomised complex intervention trial.

The risk of breast cancer patients for long-term side effects of therapy such as neurotoxicity and cardiotoxicity as well as late effects regarding comorbidities varies from individual to individual. Personalised follow-up care concepts that are tailored to individual needs and the risk of recurrences, side effects and late effects are lacking in routine care in Germany. We describe the methodology of BETTER-CARE, a parallel-arm cluster-randomised controlled trial conducted at 15 intervention and 15 control centres, aiming to recruit 1140 patients, and the results of the pilot phase. The needs- and risk-adapted complex intervention, based on existing development frameworks, includes a multidisciplinary network and digital platforms for symptom and need documentation and just-in-time adaptive interventions. The control group comprises usual care according to clinical guidelines. The primary outcome is health-related quality of life (EORTC QLQ-C30 global health), and secondary outcomes include treatment adherence. The 2-month pilot phase comprising 16 patients in one intervention and one control pilot centre demonstrated the feasibility of the BETTER-CARE approach. BETTER-CARE is a feasible intervention and study concept, investigating individualised needs- and risk-adapted breast cancer follow-up care in Germany. If successful, the approach could be implemented in German routine care. German Clinical Trial Register DRKS00028840. Registered on April 2022.

Abstract PR009: mascRNA directs nutrient-insensitive activation of the LARS-mTOR pathway in breast cancer

Abstract Cellular growth and metabolism are tightly regulated by nutrient availability, yet cancer cells frequently lose these controls, enabling unchecked proliferation even in the absence of sufficient nutrients. While non-coding RNAs are known to regulate metabolic enzyme expression, their direct role in nutrient-sensing pathways in cancer remains unclear. MALAT1-associated small cytoplasmic RNA (mascRNA) is a 61-nucleotide small RNA with a tRNA-like structure, cleaved from the 3’ end of the long non-coding RNA MALAT1. mascRNA has been shown to promote hepatocellular carcinoma proliferation and enhance protein translation in HEK293 cells, though its underlying molecular mechanism is not fully understood. We assessed mascRNA levels in over 70 human breast cancer patient samples using BaseScope assays and found that mascRNA is detectable in the cytoplasm of tumor cells, but not in normal mammary tissues or stromal cells adjacent to tumors. However, the levels of mascRNA varied significantly among patient samples, irrespective of molecular subtypes or tumor stages, suggesting that mascRNA is associated with other aspects of tumor biology. Functional studies in breast cancer cell lines revealed that mascRNA overexpression promotes cell proliferation, invasion, and metabolic pathways linked to ATP generation. Specifically, mascRNA increases basal glycolysis and mitochondrial oxidative phosphorylation and significantly enhances mitochondrial spare capacity, particularly in triple-negative breast cancer (TNBC) cell lines. We also found that mascRNA overexpression activates both mTORC1 and AMPK, indicating a deviation from the normal negative feedback loop between these two pathways. To dissect the molecular mechanism, we performed mascRNA pull-down assays and mass-spectrometric analyses. We identified RNA splicing factors, tRNA-modifying enzymes, and several aminoacyl-tRNA synthetases as mascRNA-interacting proteins. Among these, leucyl-tRNA synthetase (LARS), a component of the multi-tRNA synthetase complex, is known for its non-canonical role in activating mTORC1 at the lysosomal membrane. We found that, in MDA-MB-231 TNBC cells, mascRNA overexpression led to increased LARS localization to the lysosomal membrane where it can activate mTORC1. Interestingly, while control MDA-MB-231 cells responded to glucose and leucine availability to regulate the lysosomal localization of LARS and mTOR, mascRNA- overexpressing cells constitutively maintained LARS and mTOR localization to the lysosome, as well as mTORC1 phosphorylation, regardless of nutrient status. In summary, our data demonstrate that mascRNA aberrantly activates the LARS-mTORC1 axis in breast cancer cells independent of nutrient levels, likely through direct interaction with LARS, promoting its localization to the lysosome. This suggests that mascRNA enables breast cancer cells, and possibly other cancer types, to sustain uncontrolled growth even in nutrient-deprived conditions. Elevated mascRNA levels may thus represent a highly efficient metabolic adaptation of cancer cells. Citation Format: Bohye Park, Joshua K Stone, Steve Lim, Shuko Harada, Erin Ahn. mascRNA directs nutrient-insensitive activation of the LARS-mTOR pathway in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR009.

Abstract I004: Systematic Discovery and Annotation of Cancer Emergent Orphan non-coding RNAs in human Cancers

Abstract We recently described orphan non-coding RNAs (oncRNAs) as a class of cancer-specific small RNAs with the potential to play functional roles in breast cancer progression1. Here, we report a systematic search to annotate and characterize cancer-emergent oncRNAs across 32 tumor types. We also leverage large-scale in vivo genetic screens in xenografted mice to functionally identify driver oncRNAs in multiple tumor types. We have not only discovered a large repertoire of oncRNAs, but also found that their presence and absence represent a digital molecular barcode that faithfully captures the types and subtypes of cancer. Importantly, we discovered that this molecular barcode is partially accessible from the cell-free space as some oncRNAs are secreted by cancer cells. In a large retrospective study across 192 breast cancer patients, we showed that oncRNAs can be reliably detected in the blood and that changes in the cell-free oncRNA burden captures both short-term and long-term clinical outcomes upon completion of a neoadjuvant chemotherapy regimen. Citation Format: Hani Goodarzi. Systematic Discovery and Annotation of Cancer Emergent Orphan non-coding RNAs in human Cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I004.

Recounting the untold stories of breast cancer patient experiences: lessons learned from a patient-public involvement and engagement storytelling event.

Breast cancer remains a prevalent disease in women worldwide. Though advancements in breast cancer care have improved patient survival, a breast cancer diagnosis, and subsequent interventions have a lasting impact on patients' lived experiences during the pandemic. We present the collaborative learning process from this patient engagement workshop series as a community-academic partnership. Narrative medicine tools were used to recount patients' lived experiences following diagnosis, where both patients and researchers shared their cancer research activities in each workshop, and the role of the multidisciplinary healthcare team was discussed. We used an iterative approach to cohort building, narrative development, and the use of multiple media formats to capture stories. Over 20 patients with breast cancer shared their stories for the first time since their diagnosis with a wider audience. Here, we present the learning process and considerations from this event. Understanding patients' lived experiences can support researchers and healthcare professionals in developing an empathetic approach to shared healthcare decision making. Moreover, understanding the lived experiences of patients is critical to addressing disparities in healthcare.

Neoantigen DNA vaccines are safe, feasible, and induce neoantigen-specific immune responses in triple-negative breast cancer patients

Abstract Background Neoantigen vaccines can induce or enhance highly specific antitumor immune responses with minimal risk of autoimmunity. We have developed a neoantigen DNA vaccine platform capable of efficiently presenting both HLA class I and II epitopes and performed a phase 1 clinical trial in triple-negative breast cancer patients with persistent disease on surgical pathology following neoadjuvant chemotherapy, a patient population at high risk of disease recurrence. Methods Expressed somatic mutations were identified by tumor/normal exome sequencing and tumor RNA sequencing. The pVACtools software suite of neoantigen prediction algorithms was used to identify and prioritize cancer neoantigens and facilitate vaccine design for manufacture in an academic GMP facility. Neoantigen DNA vaccines were administered via electroporation in the adjuvant setting (i.e., following surgical removal of the primary tumor and completion of standard of care therapy). Vaccines were monitored for safety and immune responses via ELISpot, intracellular cytokine production via flow cytometry, and TCR sequencing. Results Eighteen subjects received three doses of a neoantigen DNA vaccine encoding on average 11 neoantigens per patient (range 4–20). The vaccinations were well tolerated with relatively few adverse events. Neoantigen-specific T cell responses were induced in 14/18 patients as measured by ELISpot and flow cytometry. At a median follow-up of 36 months, recurrence-free survival was 87.5% (95% CI: 72.7–100%) in the cohort of vaccinated patients. Conclusion Our study demonstrates neoantigen DNA vaccines are safe, feasible, and capable of inducing neoantigen-specific immune responses. Clinical trial registration number NCT02348320.