Biomarker For Breast Cancer Metastasis Research Articles

An Emerging Role of Long Noncoding RNAs as Novel Biomarkers for Breast Cancer Metastasis

Breast cancer is a leading cause of cancer-related deaths among women both worldwide and in Poland. Consequently, ensuring equitable access to diagnostic tests for all populations is crucial, alongside the urgent need to develop new, minimally invasive methods for early cancer detection with a particular focus on metastasis. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of cancer metastasis. This review aims to investigate the potential of lncRNAs as novel biomarkers for breast cancer, focusing on their mechanisms, clinical relevance, and therapeutic implications. A comprehensive literature search was conducted using PubMed and Google Scholar databases, targeting publications from 2013 to 2024. Keywords included “lncRNA”, “biomarker”, “breast cancer”, “metastasis”, “prognosis”, and “diagnosis”. A total of 111 articles were selected based on their relevance and quality. Recent studies have identified numerous lncRNAs such as HOTAIR, MALAT1, BANCR, NEAT1, H19, and GAS5 as key regulators of various metastatic processes in breast cancer. They can be both upregulated and downregulated. Clinical studies have shown that abnormal lncRNA expression correlates with poor prognosis, higher metastatic potential, and therapy resistance in breast cancer patients. LncRNAs have significant potential as novel biomarkers for breast cancer metastasis because of their regulatory roles in metastasis-related processes and detectability in body fluids. Further research is essential to validate these findings in larger clinical studies and to develop lncRNA-based diagnostic and therapeutic tools, ultimately improving patient outcomes in breast cancer.

Abstract PO4-13-08: Mechanical conditioning (MeCo) score is higher in circulating tumor cells (CTCs) compared to primary tumors in early-stage breast cancer and even higher in metastases compared to CTCs in metastatic breast cancer

Abstract Background: The mechanical conditioning (MeCo) score is a gene expression signature that is acquired early by cancer cells in the primary breast tumor and is reflective of their responsiveness to matrix stiffness (fibrotic-like matrix rigidity). Further, chromatin remodeling in response to stiffness allows cancer cells to retain their aggressive features even in the absence of mechanical stimulation by the primary tumor microenvironment, for instance, during their dissemination through the circulation in metastasis. Importantly, patients who have high MeCo score tumors are at higher risk of developing metastatic breast cancer, compared to low MeCo scores (p< 0.0001; HR=2.2, 95%CI 1.7-2.7; Watson et al 2021, PMID: 34192535). Moreover, circulating tumor cells (CTCs) are associated with higher rate of metastatic dissemination, making CTC detection in the circulation of breast cancer patients a significant prognostic biomarker for breast cancer metastasis. Beyond their enumeration per blood volume units, specific prognostic features of CTCs are not fully explored. We sought to determine whether, compared to primary tumors, CTCs retain high MeCo scores and whether these scores are maintained during late-stage breast cancer in the metastatic sites. Methods: CTCs were isolated from peripheral blood of two patient cohorts: stage II-III breast cancer patients using immunomagnetic enrichment/FACS methodology (Lang et al 2018, PMID: 29868978) and of metastatic breast cancer patients using ANGLE Parsortix microfluidics system (Ring et al 2022, PMID: 35000083). Gene expression profiling using RNA-seq was performed in CTCs and in matching primary tumors (PT) and metastases (MET) for the early-stage and late-stage cohorts, respectively. A quantile normalization approach was used to allow comparison across cohorts. MeCo scores were computed for all samples as per Watson et al 2021. Wilcoxon matched-pairs signed rank test was performed for comparison of MeCo scores from matching samples within each cohort; Mann-Whitney unpaired test was used to compared MeCo scores of CTCs across cohorts. Results: In 12 pairs of early-stage breast cancer patient CTCs and PT, MeCo scores of CTCs were significantly higher than their matched PTs (p=0.026). Furthermore, in 26 pairs of metastatic patient’s CTCs and MET, MeCo scores of METs were significantly higher than matching CTCs (p=0.0004). MeCo scores of CTCs were similar between early- and late-stage breast cancer despite differing CTC isolation strategies (epitope-dependent and microfluidics size gradient). 98% of genes in the MeCo score were present in the evaluable CTC, MET and PT samples. Conclusions: Our results show that the MeCo score is higher in CTCs compared to PTs and in METs compared to CTCs in early- and late-stage breast cancer, respectively. Therefore, the MeCo score is progressively higher throughout the metastatic cascade in breast cancer. These findings demonstrate that mechanical conditioning is retained during metastatic progression, even as cancer cells transition through the circulation where the initial induction by matrix stiffness is lost. Further, these findings support that cancer cells with higher MeCo scores are more competent with–and potentially selected for by–metastatic progression. Importantly, these findings provide a novel feature of CTCs, mechanical conditioning (MeCo), which is associated with higher capacity for metastasis. Further, since the CTC MeCo score is elevated even in early-stage breast cancer, it could provide, in addition to CTC enumeration, a potential prognostic tool to predict metastatic risk in breast cancer patients. Citation Format: Gus Mouneimne, Adam Watson, Adam Grant, Daniel Campo, Alexander Ring, Pushpinder Bains, Julie Lang. Mechanical conditioning (MeCo) score is higher in circulating tumor cells (CTCs) compared to primary tumors in early-stage breast cancer and even higher in metastases compared to CTCs in metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-08.

BCKDK regulates breast cancer cell adhesion and tumor metastasis by inhibiting TRIM21 ubiquitinate talin1

Breast cancer is the most common malignant cancer in women worldwide. Cancer metastasis is the major cause of cancer-related deaths. BCKDK is associated with various diseases, including proliferation, migration, and invasion in multiple types of human cancers. However, the relevance of BCKDK to the development and progression of breast cancers and its function is unclear. This study found that BCKDK was overexpressed in breast cancer, associated with poor prognosis, and implicated in tumor metastasis. The downregulation of BCKDK expression inhibited the migration of human breast cancer cells in vitro and diminished lung metastasis in vivo. BCKDK perturbed the cadherin-catenin complex at the adherens junctions (AJs) and assembled focal adhesions (FAs) onto the extracellular matrix, thereby promoting the directed migration of breast cancer cells. We observed that BCKDK acted as a conserved regulator of the ubiquitination of cytoskeletal protein talin1 and the activation of the FAK/MAPK pathway. Further studies revealed that BCKDK inhibited the binding of talin1 to E3 ubiquitin ligase-TRIM21, leading to the decreased ubiquitination/degradation of talin1. In conclusion, identifying BCKDK as a biomarker for breast cancer metastasis facilitated further research on diagnostic biomarkers. Elucidating the mechanism by which BCKDK exerted its biological effect could provide a new theoretical basis for developing new markers for breast cancer metastasis and contribute to developing new therapies for the clinical treatment of breast cancer patients.

Identification of Prognostic Biomarkers for Breast Cancer Metastasis Using Penalized Additive Hazards Regression Model.

Breast cancer (BC) has been reported as one of the most common cancers diagnosed in females throughout the world. Survival rate of BC patients is affected by metastasis. So, exploring its underlying mechanisms and identifying related biomarkers to monitor BC relapse/recurrence using new statistical methods is essential. This study investigated the high-dimensional gene-expression profiles of BC patients using penalized additive hazards regression models. A publicly available dataset related to the time to metastasis in BC patients (GSE2034) was used. There was information of 22 283 genes expression profiles related to 286 BC patients. Penalized additive hazards regression models with different penalties, including LASSO, SCAD, SICA, MCP and Elastic net were used to identify metastasis related genes. Five regression models with penalties were applied in the additive hazards model and jointly found 9 genes including SNU13, CLINT1, MAPK9, ABCC5, NKX3-1, NCOR2, COL2A1, and ZNF219. According the median of the prognostic index calculated using the regression coefficients of the penalized additive hazards model, the patients were labeled as high/low risk groups. A significant difference was detected in the survival curves of the identified groups. The selected genes were examined using validation data and were significantly associated with the hazard of metastasis. This study showed that MAPK9, NKX3-1, NCOR1, ABCC5, and CD44 are the potential recurrence and metastatic predictors in breast cancer and can be taken into account as candidates for further research in tumorigenesis, invasion, metastasis, and epithelial-mesenchymal transition of breast cancer.

Optimization of a method for the clinical detection of serum exosomal miR-940 as a potential biomarker of breast cancer

Serum exosomal microRNAs (miRNAs) are potential biomarkers for tumor diagnosis. Clinically, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) can be used to determine the expression of exosomal miRNAs in the serum of breast cancer patients. The prerequisites for obtaining meaningful serum exosomal miRNA data of breast cancer patients include a suitable extraction method for exosomes and RT-qPCR data standardized by internal reference genes. However, the appropriate methods for the extraction of exosomes and the applicability of reference genes for analyzing exosomal miRNAs in breast cancer patients remain to be studied. This study compared the effects of three exosome extraction methods as well as the expression of exosomal miRNA in different initial serum amounts and at different serum states to identify the selection of the best method for serum exosome extraction. Five candidate reference genes including miR-16, miR-484, miR-1228, miR-191 and miR-423 for standardizing serum exosomal miRNAs were screened using five algorithms and were used for the quantification of serum exosomal miR-940. Significant downregulation of serum exosomal miR-940 expression in breast cancer was detected using miR-191 and miR-1228, whereas no significant down or up regulation was observed with miR-484, miR-423 and miR-16. Previous studies have shown that the expression level of miR-940 is downregulated in breast cancer tissues. The absolute quantitative results showed that miR-940 was significantly downregulated in breast cancer serum exosomes, which was consistent with the results from the analysis using miR-191 or miR-1228 as reference genes. Therefore, miR-191 and miR-1228 could serve as reference genes for the relative quantification of serum exosomal miRNAs. This finding indicated the importance of rigorously evaluating the stability of reference genes and standardization for serum exosomal miRNA expression. Moreover, the level of serum exosomal miR-940 in breast cancer could reflect the presence of lymph node metastasis and the status of HER2/neu, which indicates its potential as a biomarker for breast cancer metastasis. In summary, an optimized protocol for the detection of serum exosomal miR-940 as a breast cancer marker was preliminarily established.

Circulating Neonatal NAV1.5 (nNAV1.5) Antigen and Anti-nNAV1.5 Antibodies as Potential Biomarkers for Breast Cancer Metastasis

Neonatal Nav1.5 (nNav1.5) has been known to potentiate breast cancer (BCa) metastasis. The detection of anti-nNav1.5 antibodies (anti-nNav1.5-Ab) reflects the immunogenicity of nNav1.5. However, the presences of circulating nNav1.5 antigen and anti-nNav1.5-Ab in the context of BCa metastasis have not been explored yet. Therefore, the study has attempted to conduct such an investigation using both blood samples from 4T1 orthotopic mice and BCa patients. In the preclinical study, forty female BALB/c mice were divided into three groups: 4T1 orthotopic BCa mice (n=17), control mice (n=20) and positive control mice (n=3). After tumour development, the mice were sacrificed to obtain target organs, whole blood, and serum. Histopathology, cytokine analyses, real-time PCR, and indirect ELISA were performed. Histopathology and cytokine analyses showed the establishment of metastasis in 4T1 orthotopic mice. The concentration of vascular endothelial growth factor (VEGF) was significantly higher in the 4T1 orthotopic mice (P<0.0001****). Circulating nNav1.5 antigen and anti-nNav1.5-Ab were detected in 4T1 orthotopic mice, using real-time PCR and indirect ELISA, respectively. Furthermore, there was an inverse relationship between anti-nNav1.5-Ab and the total metastatic foci (P=0.0485*, r=-0.7306). In the clinical study, 32 BCa patients were grouped based on their stages: early-invasive (n=15) and advanced (n=17) stages. Approximately 3 mL of blood was withdrawn, and only indirect ELISA was conducted. The clinical study showed that BCa patients of advanced-stages portrayed higher expression of anti-nNav1.5-Ab compared to early stages of BCa (P =0.0110*). In conclusion, the detection of nNav1.5 antigen and anti-nNav1.5-Ab was consistent with the presence of BCa metastasis.

TAp63 regulates bone remodeling by modulating the expression of TNFRSF11B/Osteoprotegerin

ABSTRACT The transcription factor p53 has been shown to control the differentiation process of the mesenchymal stem cells (MSCs). As a matter of fact, in vivo p53 loss leads to an unbalance between bone formation versus bone erosion. Using as experimental system, human bone marrow-derived MSCs and mouse bone marrow-derived TAp63-/- MSCs, we have asked whether the other members of the p53 family, p63 and p73, are involved in controlling MSCs osteogenic differentiation. Our results indicate that both during human and mouse MSC-induced osteogenic differentiation, TAp63 isoforms are mainly upregulated in comparison with p73 isoforms. In addition, MSCs derived from TAp63 knock-out mice show delayed osteogenic differentiation. Interestingly, we found that, in contrast to p53, TAp63 trascriptionally regulates osteoprotegerin (OPG or TNFRSF11B) important for bone remodeling and osteoclastogenesis inhibition throughout the RANKL/RANK/OPG pathway. Analysis of the expression of p63 and OPG in breast cancer, which is one of the most common human cancers that metastatizes to bone, showed that p63/OPG pathway is deregulated and that a higher expression of both p63 and OPG is associated with a better patient survival, suggesting that p63/OPG axis may be further investigated as clinical biomarker for breast cancer metastasis. Abbreviations MSC, mesenchymal stem cells; OPG, osteoprotegerin; RUNX2, Run-trelated transcription factor 2

MiR-128 suppresses metastatic capacity by targeting metadherin in breast cancer cells

BackgroundBreast cancer, the most common cancer in women worldwide, causes the vast majority of cancer-related deaths. Undoubtedly, tumor metastasis and recurrence are responsible for more than 90 percent of these deaths. MicroRNAs are endogenous noncoding RNAs that have been integrated into almost all the physiological and pathological processes, including metastasis. In the present study, the role of miR-128 in breast cancer was investigated.ResultsCompared to the corresponding adjacent normal tissue, the expression of miR-128 was significantly suppressed in human breast cancer specimens. More importantly, its expression level was reversely correlated to histological grade of the cancer. Ectopic expression of miR-128 in the aggressive breast cancer cell line MDA-MB-231 could inhibit cell motility and invasive capacity remarkably. Afterwards, Metadherin (MTDH), also known as AEG-1 (Astrocyte Elevated Gene 1) and Lyric that implicated in various aspects of cancer progression and metastasis, was further identified as a direct target gene of miR-128 and its expression level was up-regulated in clinical samples as expected. Moreover, knockdown of MTDH in MDA-MB-231 cells obviously impaired the migration and invasion capabilities, whereas re-expression of MTDH abrogated the suppressive effect caused by miR-128.ConclusionsOverall, these findings demonstrate that miR-128 could serve as a novel biomarker for breast cancer metastasis and a potent target for treatment in the future.

TIPE3 protein promotes breast cancer metastasis through activating AKT and NF-κB signaling pathways.

TIPE3 (TNFAIP8L3) is the transfer protein of phosphoinositide second messengers that promote cancer. Its role in breast cancer has not been evaluated. We report here that TIPE3 protein was significantly upregulated in human breast cancer tissues as compared with adjacent non-tumor tissues from the same patients. The level of TIPE3 protein in invasive ductal carcinoma was significant higher than that in ductal carcinoma in situ (DCIS), and the level of TIPE3 in lymphatic metastasized carcinoma was higher than that in invasive ductal carcinoma from the same patients. Additionally, the level of TIPE3 protein was positively correlated with the level of human epidermal growth factor receptor 2 (HER-2), and TIPE3 expression was significantly higher in high-invasive breast cancer cell lines than that in low-invasive cell lines. Importantly, TIPE3 knockdown in breast cancer cells inhibited cell proliferation, migration, and invasion in vitro, whereas TIPE3 overexpression had the opposite effect. In mice, TIPE3 expression significantly promoted the metastasis of breast cancer cells. TIPE3 expression also increased the level of MMP2 and uPA, and the activation of the AKT and NF-κB signaling pathways. These results demonstrate that TIPE3 may promote breast cancer growth and metastasis through AKT and NF-κB, and may serve as a potential biomarker for breast cancer metastasis.

Identification of Nucleobindin-2 as a Potential Biomarker for Breast Cancer Metastasis Using iTRAQ-based Quantitative Proteomic Analysis.

Metastasis is a lethal step in the progression of breast cancer. None of the metastasis-associated biomarkers identified up to now has a definite prognostic value in breast cancer patients. This study was designed to identify biomarkers for breast cancer metastasis and predictors of the prognosis of breast cancer patients. The differentially expressed proteins between 23 paired primary breast tumor and metastatic lymph nodes were identified by quantitative iTRAQ proteomic analysis. Immunohistochemistry was applied to locate and assess the expression of NUCB2 in paired primary breast tumor and metastatic lymph node tissues (n = 106). The relationship between NUCB2 expression and the clinicopathological characteristics of breast cancer patients (n = 189) were analyzed by χ2 test. Kaplan-Meier analysis and Cox hazard regression analysis were utilized to investigate the relationship between its expression and prognosis of breast cancer patients. The iTRAQ proteomic results showed that 4,837 confidential proteins were identified, 643 of which were differentially expressed in the primary breast cancer tissues and the paired metastatic lymph nodes. NUCB2 protein was found decreased in paired metastatic lymph nodes (P = 0.000), with the positive expression rate being 82% in primary breast cancer tissues and 47% in paired metastatic lymph nodes, respectively. According to Kaplan-Meier analysis, the overall survival time of patients with positive expression of NUCB2 protein were shorter than those with negative NUCB2 expression (P = 0.004). Cox regression model suggested that NUCB2 was a risk factor of breast cancer patients (P = 0.045, RR = 1.854). We conclude that NUCB2 can be used as a potential biomarker for breast cancer metastasis and a prognostic predictor of breast cancer patients.

DNA aptamer probes for detection of estrogen receptor α positive carcinomas

Estrogen receptor alpha (ERα) also known as NR3A1 (nuclear receptor subfamily 3, group A, member 1) is a ligand-activated transcription factor. It is an important biomarker for breast cancer metastasis. In the present study, we report a novel DNA aptamer candidate against estrogen receptor (ER) alpha structure. Theenriched aptamercandidate was obtained after 14 iterative cycles of invitroprotein-SELEX process. Isothermal calorimetry study suggests the nanomolar sensitivity of the candidate ER_Apt1 to its target protein. Fluorescence- and chemiluminescence-binding assays confirm the specificity of the candidate aptamer to ER alpha positive breast cancer cell line. Comparative analysis of ER_Apt1 to ER alpha monoclonal antibody wasalso performed to analyze the expression of ER alpha in various malignant cancer cell line. Cytochemical and immunohistochemistry assay indicates its potential use as a diagnostic agent against ERα positive carcinomas. The nucleotide aptamer sequences described in the present study can be used for the detection, treatment, prophylaxis and diagnosis of ERα-related disorder.

MicroRNA-376b promotes breast cancer metastasis by targeting Hoxd10 directly.

Breast cancer is the most common malignant disease in women, and metastasis formed at distant anatomic sites was the major cause of cancer-related mortality. Thus, a novel therapy target and progression biomarker for breast cancer metastasis was necessary. microRNA (miR)-376b has been demonstrated to regulate angiogenesis; however, its role in cancer metastasis remains elusive. In the present study, the expression of miR-376b in normal breast tissue, JC and 4T1 cells was determined by qPCR. Furthermore, in vitro and in vivo experiments were performed to determine the effect of miR-376b on breast cancer metastasis. The direct target of miR-376b was determined by the luciferase assay and western blotting. The results indicated that silencing of miR-376b by the miR-376-mimic significantly inhibited 4T1 cell migration and invasion in vitro. Lung metastasis was also evidently decreased after silencing of miR-376b in 4T1 cells. Moreover, the luciferase assay and western blotting identified that Hoxd10 is the direct target of miR-376b during the regulation of breast cancer metastasis. To the best of our knowledge, the present study was the first to demonstrate the promoting breast cancer metastasis role of miR-376b by directly targeting Hoxd10. Therefore, it would be a novel therapy target and prognostic biomarker for breast cancer.

Detecting Pairwise Interactive Effects of Continuous Random Variables for Biomarker Identification with Small Sample Size.

Aberrant changes to interactions among cellular components have been conjectured to be potential causes of abnormalities in cellular functions. By systematic analysis of high-throughput-omics data, researchers hope to detect potential associations among measured variables for better biomarker identification and phenotype prediction. In this paper, we focus on the methods to measure pairwise interactive effects among continuous random variables, representing molecular expressions, with respect to a given categorical outcome. Together with a comprehensive review on the existing measures, we further propose new measures that better estimate interactive effects, especially in small sample size scenarios. We first evaluate the performance of the existing and new methods for both small and large sample sizes based on simulated datasets that shows our proposed methods outperform previous methods in general. The best performing method for small sample size scenarios suggested by simulation experiments is then implemented to estimate interactive effects among genes with respect to the metastasis outcome in two breast cancer studies based on micro-array gene expression datasets. Our results further demonstrate that integrating detected interactive effects together with individual effects can help in finding more accurate biomarkers for breast cancer metastasis, which are indeed involved in important pathways related to cancer metastasis based on gene set enrichment analysis.

MiR-340 suppresses cell migration and invasion by targeting MYO10 in breast cancer.

Breast cancer is one of the most common malignant tumors among females, and can seriously affect the physical and mental health and even threaten the lives of women. Recently, research has demonstrated that microRNAs (miRNAs), as a new method of regulation, have been shown to have oncogenic and tumor‑suppressive functions in human breast cancer. Detection of their expression may lead to the identification of novel markers for breast cancer. In the present study, we firstly detected miR‑340 expression and found lower expression of miR‑340 in 6 human breast cancer cell lines by using RT‑qPCR. Then by using wound healing assay and Transwell migration and invasion experiments, we focused on the role of miR-340 in the regulation of tumor cell migration and invasion, exploring the relationship between them. The results revealed that induction of miR‑340 expression was able to suppress tumor cell migration and invasion, whereas knockdown of miR‑340 expression promoted breast cancer cell migration and invasion. At the gene level, MYO10 (myosin X), as a direct miR‑340 target gene, mediated the cell migration and invasion. Finally, we verified our research further at the tissue specimen level and in animal experiments. In brief, miR‑340 plays an important role in breast cancer progression. Thus, miR‑340 may be further explored as a novel biomarker for breast cancer metastasis and prognosis, and potentially a therapeutic target.

TGF-β upregulates miR-181a expression to promote breast cancer metastasis

Late-stage breast cancer metastasis is driven by dysregulated TGF-β signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-β. Here we establish miR-181a as a TGF-β-regulated "metastamir" that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer.

Detection of prolactin inducible protein mRNA, a biomarker for breast cancer metastasis, using a molecular beacon-based assay

Mortality due to breast cancer is increasingly linked to early, undetected metastasis, making methods for earlier detection acutely necessary. We describe the development of an assay based on molecular beacon (MB) chemistry with fluorescence detection to monitor a breast cancer biomarker for the analysis of breast cancer metastasis. The MB assay is based on the complementary base-pairing interactions of the MB nucleic acid with mRNA indicative of breast cancer metastasis. The presence of mRNA is characterized by an increase in the fluorescence intensity of the molecular beacon. The assay gives a linear, reproducible response to prolactin inducible protein mRNA, with a limit of detection in the high picomolar range. This method sensitively and specifically identifies a biomarker directly in serum samples in minimal time and with a straightforward procedure, dramatically reducing the total time for sample analysis over current methods from days to hours. The potential impact of this work in detection and understanding of breast cancer metastasis lies in improvements in simplicity, accuracy, and speed over current methods, which could allow for improved patient treatment and prognoses. Ultimately, additional sample throughput will result in better understanding of disease progression.

Abstract 3305: The role of carbonic anhydrase IX in tumor hypoxia, metastasis and the cancer stem cell niche

Abstract Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1 inducible protein that regulates intra- and extracellular pH under hypoxic conditions, and promotes tumor cell survival and invasion in hypoxic microenvironments. shRNA-mediated depletion of CAIX expression in mouse and human metastatic breast cancer cells, capable of inducing CAIX in hypoxia, resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. CAIX depletion led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. In addition, the CD44+/CD24-/low “cancer stem cell” compartment, which exhibited hallmarks of EMT (E-cadherin low/SMA high) was significantly enhanced in hypoxia, but was severely depleted in shRNA-CAIX knock-down cells as well as in cells treated with CAIX small molecule inhibitors. CAIX depletion or inhibition of its activity prevented mesenchymal conversion of the stem cells and also resulted in the suppression of expression of several EMT and “stemness” regulator genes. Treatment of mice harboring CAIX-positive 4T1 and MDA-MB-231/LM2 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without any inhibitory effects on CAIX negative tumors, or any adverse toxic effects. This was associated with significant depletion of cells expressing “cancer stem cell” markers. Furthermore, interrogation of 3,630 human breast cancers revealed CAIX to be an independent poor prognostic biomarker for distant metastases and survival. Our findings demonstrate that CAIX is vital for the survival of “cancer stem cells” in hypoxic niches as well as for the growth and metastasis of hypoxic breast tumors. It is a specific, targetable biomarker for breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3305. doi:1538-7445.AM2012-3305

Targeting Tumor Hypoxia: Suppression of Breast Tumor Growth and Metastasis by Novel Carbonic Anhydrase IX Inhibitors

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.

Abstract 5270: Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer

Abstract Histone deacetylases (HDACs) are associated with the development and progression of cancer, but which HDAC isoforms play important roles in breast cancer metastasis is not known. Here, we identify the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) than MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5270. doi:10.1158/1538-7445.AM2011-5270

Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.