Role In Breast Cancer Recurrence Research Articles

Abstract 807: Inflammatory biomarkers: Breast cancer survival and possible role in COVID-19 associated comorbidity

Abstract It is known that inflammatory cytokines exacerbate the persistence and severity of various disease states. Breast cancer is the most frequently detected cancer among women worldwide and our recent studies suggest that the inflammatory state of breast (BrCa) cancer, a byproduct of elevated cytokine expression, induces epigenetic modifications leading to increased recurrence. Ongoing NCI clinical trial data (ClinicalTrials.gov, CCC19, NCT04354701) indicates that among patients with cancer and COVID-19, the mortality is high, and the most prevalent malignancies are of breast [21%] and prostate [16%] origin. Due to the risk of cytokine storm during SARS-CoV-2 infection, it is crucial to identify potential mechanisms of hyperinflammation in BrCa patients. In this study, we have evaluated the level of copy number alteration (CNA) of different inflammatory cytokines including IL-8, IL-1b, IL6, IL-8, GM-CSF, TNF-alpha and many others using cBioportal platform which includes over sixty-nine thousand tumor samples (n>69,000 from 213 different studies) from over 33 different cancers. We found that IL-8 has the highest level of amplification in different breast cancers subtypes. Besides, we also analyzed serum samples from BrCa patients, both recurrent and non-recurrent, by different proteomics methods to identify serum cytokines involved in prognosis and recurrence. Comparative data analysis between non-recurrent BrCa against recurrent BrCa patients identified several proteins with very high significance, mostly proteins associated with epigenetic pathways including HDAC9 (P = 0.0035), HDAC5 (P = 0.013), and HDAC7 (P = 0.020). Besides, we identified differential expression of several pro-inflammatory cytokines and immune regulators (IL-8, IL-4, IL-18, IL-12p70) that were present only in recurrent BrCa patient serum. Our data indicate that inflammatory processes contribute to epigenetic modifications that ultimately play a critical role in breast cancer recurrence. In terms of COVID-19 associated co-morbidity, the already dysregulated inflammatory state of BrCa patients may increase their susceptibility to cytokine-storm, leading to increased severity of COVID-related complications and increased mortality rate. Specifically, we hypothesize that the identified elevated level of IL-8 in BrCa patients may lead to a higher basal level of inflammation and contribute to the risk of attaining cytokine-storm during SARS-CoV-2 infection, making it a valuable target for future studies. Citation Format: Alakesh Bera, Eric Russ, Surya Radhakrishnan, Hai Hu, COL Craig D. Shriver, Meera Srivastava. Inflammatory biomarkers: Breast cancer survival and possible role in COVID-19 associated comorbidity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 807.

Proteomic Analysis of Inflammatory Biomarkers Associated With Breast Cancer Recurrence.

Breast cancer is the most frequent cancer detected for women, and while our ability to treat breast cancer has improved substantially over the years, recurrence remains a major obstacle. Standard screening for new and recurrent breast cancer involves clinical breast imaging. However, there is no clinically approved noninvasive body fluid test for the early detection of recurrent breast cancer. Materials and Method: In this study, we analyzed serum samples from both recurrent and nonrecurrent breast cancer patients by different proteomics methods to identify biomarkers in patients with recurrence of disease. Comparative data analysis identified several histone deacetylase (HDAC) proteins, which were found at significantly higher levels in the serum of recurrent breast cancer patients: HDAC9 (C-term) (P=0.0035), HDAC5 (C-term) (P=0.013), small ubiquitin-like modifier 1 (N-term) (P=0.017), embryonic stem cell-expressed Ras (inter) (P=0.018), and HDAC7 (C-term) (P=0.020). Chronic inflammation plays a critical role in the development of the breast cancer recurrence, and we identified several proinflammatory cytokines that were present at elevated levels only in recurrent breast cancer patient serum. Our data indicated that the epigenetic regulation of inflammatory processes plays a critical role in breast cancer recurrence. The identified proteins could lay the groundwork for the development of a serum-based breast cancer recurrence assay.

Abstract 3931: The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment

Abstract Triple negative breast cancer (TNBC) recurrence rates remain high despite aggressive therapeutic intervention, including surgery, chemotherapy, and radiotherapy (RT). Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence rather than persistent tumor cells in the irradiated surgical bed. However, the role of the microenvironment in recurrence is not well understood. An emerging risk factor for breast cancer patients is lymphopenia or abnormally low systemic lymphocyte counts following therapy. In this study, we examined whether lymphopenia following therapy correlates to local recurrence. To investigate the relationship between prolonged low lymphocyte count and recurrence, we evaluated radiation effects on tumor and immune cell recruitment to normal tissues using an orthotopic breast cancer model of lymphopenia. We tested the hypothesis that local recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. Local cytokine secretion as well as protein expression levels were analyzed to evaluate how tumor-stromal interactions modulate tumor cell recruitment to sites of damage. Recurrence free survival at 5 years for TNBC patients with sustained low ALC 1-5 months after RT (n=37) was 62.5% compared with 97% for patients with normal ALC (n=46; p<0.0001). Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the mammary fat pad (MFP) and surrounding tissues in BALB/c mice with depleted CD8+ T cells. Macrophage infiltration was greatly enhanced in BALB/c mice with depleted CD8+ T cells (p<0.0001) and necessary for tumor cell recruitment. Luminex analysis of MFPs in mice lacking CD8+ T cells after RT showed enhanced cytokine secretion of factors that regulate the inflammatory microenvironment and influence tumor cell proliferation and invasion. RPPA analysis of irradiated MFPs in the absence of CD8+ T cells revealed factors that promote epithelial-mesenchymal transition. Taken together, these results demonstrate that the response of the tissue microenvironment to therapy is dependent on the immune milieu and may encourage local recurrence. Our study establishes the importance of considering tumor subtype and immune function when assessing failure and outcome risks in breast cancer. We show that normal tissue radiation response may play a role in modulating tumor and immune cell migration. These results suggest that the stroma facilitates local recurrence in breast cancer through influencing immune and tumor cell behavior following RT. Citation Format: Benjamin C. Hacker, Steven M. Alves, Dadi Jiang, Albert C. Koong, Amato J. Giaccia, Edward E. Graves, Marjan Rafat. The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3931.

MiR-106b-5p and miR-17-5p could predict recurrence and progression in breast ductal carcinoma in situ based on the transforming growth factor-beta pathway

PurposeDuctal carcinoma in situ (DCIS) is well-known precursor of invasive ductal carcinoma (IDC). Parts of patients show recurrence as DCIS or IDC after local treatment, but there are no established markers predicting relapse. We analyzed changes in miRNA and oncogene expression during DCIS progression/evolution to identify potential markers predicting recurrence.MethodsForty archival tissues diagnosed as primary or recurrent DCIS and DCIS adjacent to IDC were analyzed. MiRNA hierarchical clustering showed up-regulation of miR-17-5p and miR-106b-5p in recurrent DCIS and DCIS adjacent to IDC. Target genes were predicted based on pre-formed miRNA databases and PanCancer Pathway panel. MiRNAs were transfected into MCF-10A and MCF-7 cells; western blot analysis was performed with MCF-7 cell line to evaluate the effects on TGF-β downstream pathway.ResultsmiRNA hierarchical clustering showed 17 dysregulated miRNAs, including miR-17-5p and miR-106b-5p. Based on miRNA database and nCounter Pancancer pathway analysis, TGFβRII was selected as target of miR-106b-5p and miR-17-5p. MiR-106b-5p- and miR-17-5p-transfected MCF-7 cells showed decreased expression of TGFβRII, especially in cells transfected with both miRNAs.ConclusionmiR-106b-5p and miR-17-5p might have a role in breast cancer recurrence and progression by suppressing TGF-β activity, leading to early breast cancer carcinogenesis.

Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways.

The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer.

A Novel Role of Primary Cilia in Breast Cancer Recurrence

Recurrent tumors are the predominant cause of breast cancer‐related deaths, therefore there is an urgent need for more effective therapeutic strategies. However, a critical barrier to their development is a lack of understanding of the molecular mechanisms involved in breast tumor recurrence. Recently, primary cilia, microtubule‐based organelles that project from the surface of vertebrate cells, have emerged into the spotlight for their link to numerous human diseases, including cancer. However, the relationship between primary cilia and breast cancer recurrence remains unknown. Here, using a genetically engineered mouse model of breast cancer, which closely recapitulates the clinical phenomenon of breast cancer recurrence, we have uncovered that primary cilia are more highly enriched in recurrent mammary tumors than in both primary mammary tumors and asymptomatic/contralateral mouse mammary tissues. Congruently, we found that the percentage of ciliated cells was substantially elevated in recurrent breast cancer cells compared to primary breast cancer cells. Moreover, RNA‐sequence analysis also revealed that GLI2 and GLI3, which are major downstream effectors of the morphogenic Sonic Hedgehog signaling pathway and are also well‐known to localize to primary cilia, were upregulated in recurrent breast cancer cells compared to primary breast cancer cells. Collectively, these data suggest that primary cilia may have a role in breast cancer recurrence. Our study also indicates that, at least within the recurrent stages of the disease, the primary cilium may serve as a specialized oncogenic signaling organelle.Support or Funding InformationTo Sarah C. Goetz: R00 HD076444 (from the National Institute of Child Health and Human Development).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a FFQ for breast cancer survivors in Korea.

Diet may play an important role in breast cancer recurrence or survival, and therefore assessment of long-term diet among breast cancer survivors is important in breast cancer survivorship research. Given that the diet of breast cancer survivors may differ from that of the general population, the use of a FFQ specific to this group may be needed. The objective of this study was to develop a FFQ for breast cancer survivors, the most commonly used tool to measure long-term dietary patterns in nutritional epidemiological studies. We collected information on the foods and amounts of foods consumed using 3-d dietary records from a total of 192 women who had been diagnosed with stage I-III breast cancers and had undergone breast cancer surgery at least 6 months before the baseline study. A total of 1254 foods and dishes consumed were re-grouped by the similarity of the main ingredients and/or serving units, and several dishes commonly consumed among the Korean population were added. After we performed contribution analyses and variability analyses to detect between-person variation for selected nutrients, we listed a total of 123 foods and dishes for the FFQ specific to breast cancer survivors. Our breast cancer survivor-specific FFQ can be used to estimate long-term dietary intake and to examine its association with breast cancer prognosis in epidemiological studies of breast cancer in Korea.

Abstract P5-04-15: Investigating the role of the AMPK signaling cascade in breast cancer tumor dormancy

Abstract Breast cancer can recur in patients long after treatment for initial disease. The mechanisms underlying recurrence as well as the characteristics of these dormant tumor cells remain poorly understood. AMPK (AMP-activated protein kinase) is the central metabolic regulator of the cell, and has two catalytic isoforms, a1 and α2. The loss of AMPK, more specifically AMPKα2, is observed in primary breast cancer and its expression could function to limit tumor expansion in vivo. We hypothesize that the expression of AMPKα2 will confer a survival advantage in dormant breast cancer cells by upregulating the p38MAPK signaling axis. In order to investigate breast cancer dormancy mechanisms in vitro, we generated mammospheres using the estrogen-dependent MCF-7 human breast cancer cell line, which has low expression of AMPKα2. Control MCF-7 cells expressing GFP and MCF-7 cells overexpressing the AMPKα1 or AMPKα2 isoforms were cultured as mammospheres and exposed to various metabolic stressors including estrogen, glucose, and serum deprivation. Spheroids were evaluated for size, viability, gene expression at the RNA level by qRT-PCR, and intracellular signaling by immunoblot as well as immunohistochemistry. AMPKα2 expressing mammospheres were larger and more viable than either the GFP or AMPKα1 expressing spheres. In addition, these cells were resistant to serum deprivation, and upregulated members of the p38MAPK signaling axis, a key player in tumor dormancy. To evaluate estrogen deprivation-induced dormancy in vivo, MCF-7 cell lines expressing either GFP or AMPKα2 embedded in a matrigel matrix were implanted in athymic nude mice containing supplemental slow-release estradiol pellets. After one week, estradiol pellets were removed to induce a dormant period for roughly thirty days. Evaluation of matrigel plugs at this time indicated that AMPKα2 expressing cells demonstrated significant survival whereas GFP expressing cells were significantly depleted. Estadiol pellets were then re-implanted for five weeks to determine if the AMPKα2 expressing cells still responded to estradiol supplementation.This resulted in the AMPKα2 expressing tumors rapidly expanding to a maximal size as compared to the GFP expressing tumors. Consistent with our findings in vitro using mammosphere cultures, the AMPKα2 expressing cells had higher levels of phospho-p38MAPK than the GFP expressing cells, as seen by immunofluorescence. We conclude that the expression of AMPKα2 promotes both survival and dormancy in estrogen-sensitive breast tumor cells, thus implying a significant role in breast cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-15.

Abstract A32: Notch signaling regulates recurrence in a model of HER2 targeted therapy

Abstract Breast cancer is the leading cause of cancer-related deaths among women worldwide. While mortality rates have improved over the past thirty years, physicians still lack effective tools for the prevention or treatment of breast cancer recurrence. An essential first step in designing such therapies will be to elucidate the pathways that contribute to therapeutic resistance, dormancy, and recurrence. Our laboratory has developed a series of mouse models that permit the conditional activation of oncogenes in the mammary glands of mice and can be used to recapitulate key features of breast cancer progression including dormancy and recurrence after targeted therapy. In the MMTV-rtTA;TetO-neu (MTB/TAN) model, treatment with doxycycline (dox) permits mammary specific activation of HER2/neu and drives primary tumor formation. Upon removal of dox and resultant down-regulation of HER2/neu, primary tumors regress as a consequence of oncogene addiction. However, a small population of tumor cells persists in a histologically identifiable residual lesion. After a period of cellular dormancy, residual tumor cells reenter the cell cycle in a stochastic manner and give rise to recurrent tumors, independent of HER2/neu signaling. Recent evidence suggests that Notch signaling may play a role in breast cancer recurrence. For example, elevated Notch signaling in ductal carcinoma in situ lesions has been found to be associated with early recurrence after surgery. To build on these data, we analyzed microarray datasets from breast cancer patients and found that NOTCH1 expression levels are correlated with recurrence risk, suggesting that this pathway may play a role in human disease. Furthermore, in our mouse model system, we have found that Notch signaling is activated following HER2/neu downregulation. To assess the functional significance of this phenomenon, we performed in vitro clonogenic assays and in vivo recurrence assays. Activation of Notch signaling accelerated colony formation and recurrence, whereas Notch repression inhibited colony formation and recurrence. These data suggest that Notch signaling is both necessary and sufficient to promote recurrence following HER2/neu downregulation. Taken together, our data support a model in which the Notch signaling pathway provides a mechanism by which HER2/neu driven tumors can escape therapy and cause recurrent disease. As gamma secretase inhibitors are currently in late-stage clinical trials for the treatment of breast cancer, work to determine whether Notch signaling contributes to the survival and recurrence of breast cancer cells could have a significant clinical impact. The observation that Notch signaling is required for recurrence would raise the possibility that these drugs could be used to target residual tumor cells and prevent breast cancer recurrence.

Interaction of dietary polyphenols with molecular signaling pathways of antiestrogen resistance: possible role in breast cancer recurrence.

Abstract Breast cancer is the most common cancer diagnosed in women and its global incidence is rising rapidly. Adjuvant hormonal therapy, with antiestrogens (AE) such as tamoxifen and fulvestrant, is highly effective in the treatment of estrogen receptor-positive (ER+) breast cancers and is largely responsible for the increase in survival rates seen in the past four decades. However, nearly 50% of women with ER+ cancer display de novo or acquired resistance to AE therapies. Potential molecular mechanisms driving the resistance phenotype are beginning to be elucidated, allowing further development of more effective therapeutic and preventive strategies to reduce the overall mortality due to breast cancer. Over 70% of breast cancer survivors surveyed report increasing their comsumption of fruits, vegetables, and natural product supplements upon diagnosis. These are rich sources of dietary polyphenols (PPs) that can interact with cell-signaling pathways involved in the development of AE resistance. However, research on mechanisms by which these agents may affect AE resistance and whether PP intake can significantly change breast cancer recurrence is limited. We summarize the available data on the effects of PPs on breast cancer recurrence and the interactions of these compounds with some of the signaling pathways hypothesized to drive cell death and survival involved in the development of AE resistance in breast cancer.