Metastatic Progression Of Breast Cancer Research Articles

Abstract 4016: Serum proteomics uncover possible biomarkers associated with progression of metastatic breast cancer

Abstract Despite the recognized advances in the treatment of breast cancer, it still accounts for 15% of all cancer-related deaths. Ninety percent of breast cancer deaths are due to unpredicted metastasis. There is neither successful treatment for metastatic patients nor a specific test to predict or detect secondary lesions. Patients with primary tumor will be either overtreated with cytotoxic side effects or undertreated and risk recurrence. This necessitates the need for personalized treatment, which is hard to offer for such heterogeneous disease. Obstacles in treating breast cancer metastasis are mainly due to the gaps exist in the understanding of the molecular mechanism of metastasis. The linear model of metastasis is supported by several observations that reflect an early crosstalk between the primary and secondary tumor, which in turn makes the secondary microenvironment fertile for the growth of disseminated cells. This communication occurs through circulation and utilizes molecules which have not been identified to date. Identifying such molecules may help in detecting initial stages of tumor colonization and predict the target organ of metastasis. Furthermore, these molecules may help to provide a personalized therapy that aims to tailor treatment according to the biology of the individual tumor. Advances in proteomics allows for more reproducible and sensitive biomarker discovery. Proteomic biomarkers are often more translatable to the clinic compared to biomarkers identified using other -omics approaches. Further, protein biomarkers can be found in biological fluids making them a non invasive way to treat or investigate cancer patients. In this study we are screening serum samples of stage I and stage III breast cancer patients using cLC-MS/MS, with a focus on characterizing the differential expression of small peptide biomarkers. We typically analyze 2500 individual small peptide biomarkers in a single serum sample. Comparisons between different sample types (from stage I and III breast cancer patients in this case) allows for the detection of unique short peptide biomarkers present in one sample type. In our preliminary analysis of 6 patients of each type, we find roughly 64 short peptide biomarkers to be upregulated in patients with metastatic breast cancer. We also see the downregulation of an addition 92 short peptides in these same patients. The serum expression pattern and identity of peptides in metastatic patients may provide clues about the biology underlying tumor cell dissemination and colonization to specific sites. Citation Format: Adhari A. AlZaabi, Marc Hansen, Steven Graves. Serum proteomics uncover possible biomarkers associated with progression of metastatic breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4016. doi:10.1158/1538-7445.AM2014-4016

Association of vascular endothelial growth factor – A gene polymorphisms and haplotypes with breast cancer metastases

Background. Vascular endothelial growth factor (VEGF-A) is a key regulator of tumor-induced angiogenesis and essential for tumor growth and distant tumor spread. The aim of the present study was to evaluate the role of VEGF-A polymorphisms and haplotypes for metastatic progression in breast cancer patients.Material and methods. We performed a prospective study including 801 breast cancer patients. Occurrence of metastases was examined in regular follow-up investigations. Seven VEGF-A polymorphisms were selected and determined by 5′-nuclease assays (TaqMan). The selection of VEGF-A variants was based upon their location (promoter or UTR) as well as a minor allele frequency of at least 0.10. Haplotypes and linkage disequilibrium were determined using the Haploview program.Results. Within a median follow-up time of 84 months, 165 (21%) patients developed distant metastases. In univariate analysis, carriers of the CCCCC haplotype formed by five polymorphisms upstream the coding region were at decreased risk of distant metastases [hazard ratio (HR) = 0.743; 95% CI 0.579–0.953; p = 0.019]. Univariate analysis also revealed a decreased risk of distant metastases for postmenopausal patients carrying the -634G> C polymorphism (HR 0.704; 95% CI 0.514–0.965; p = 0.029) and the CCCCC haplotype (HR = 0.645; 95% CI 0.464–0.898; p = 0.009). After adjustment for other co-variates, the HR for distant metastases was 0.651 (95% CI 0.447–0.948) for postmenopausal carriers of the -634G> C polymorphism (p = 0.025; corrected p-value = 0.262), and 0.586 (95% CI 0.393–0.873) for postmenopausal patients with the CCCCC haplotype (p = 0.009, corrected p-value = 0.189).Conclusion. The results from univariate and multivariate analyses suggest an influence of VEGF-A gene variants on the development of distant metastases in breast cancer patients. However, none of the observed associations reached statistical significance after correction for the effects of multiple testing. Additional prospective and sufficiently powered studies are essential before firm conclusions about the role of VEGF-A gene variants for distant progression in breast cancer can be drawn.

NMI and EMT.

NMI and EMT.

Fragment N2, a caspase‐3‐generated RasGAP fragment, inhibits breast cancer metastatic progression

The p120 RasGAP protein negatively regulates Ras via its GAP domain. RasGAP carries several other domains that modulate several signaling molecules such as Rho. RasGAP is also a caspase-3 substrate. One of the caspase-3-generated RasGAP fragments, corresponding to amino acids 158-455 and called fragment N2, was previously reported to specifically sensitize cancer cells to death induced by various anticancer agents. Here, we show that fragment N2 inhibits migration in vitro and that it impairs metastatic progression of breast cancer to the lung. Hence, stress-activated caspase-3 might contribute to the suppression of metastasis through the generation of fragment N2. These results indicate that the activity borne by fragment N2 has a potential therapeutic relevance to counteract the metastatic process.

Genetic and Phenotypic Diversity in Breast Tumor Metastases

Metastatic disease is the main cause of cancer-related mortality due to almost universal therapeutic resistance. Despite its high clinical relevance, our knowledge of how cancer cell populations change during metastatic progression is limited. Here, we investigated intratumor genetic and phenotypic heterogeneity during metastatic progression of breast cancer. We analyzed cellular genotypes and phenotypes at the single cell level by performing immunoFISH in intact tissue sections of distant metastatic tumors from rapid autopsy cases and from primary tumors and matched lymph node metastases collected before systemic therapy. We calculated the Shannon index of intratumor diversity in all cancer cells and within phenotypically distinct cell populations. We found that the extent of intratumor genetic diversity was similar regardless of the chromosomal region analyzed, implying that it may reflect an inherent property of the tumors. We observed that genetic diversity was highest in distant metastases and was generally concordant across lesions within the same patient, whereas treatment-naïve primary tumors and matched lymph node metastases were frequently genetically more divergent. In contrast, cellular phenotypes were more discordant between distant metastases than primary tumors and matched lymph node metastases. Diversity for 8q24 was consistently higher in HER2(+) tumors compared with other subtypes and in metastases of triple-negative tumors relative to primary sites. We conclude that our integrative method that couples ecologic models with experimental data in human tissue samples could be used for the improved prognostication of patients with cancer and for the design of more effective therapies for progressive disease.

Inhibiting Metastatic Breast Cancer Cell Migration via the Synergy of Targeted, pH-triggered siRNA Delivery and Chemokine Axis Blockade

Because breast cancer patient survival inversely correlates with metastasis, we engineered vehicles to inhibit both the C-X-C chemokine receptor type 4 (CXCR4) and lipocalin-2 (Lcn2) mediated migratory pathways. pH-responsive liposomes were designed to protect and trigger the release of Lcn2 siRNA. Liposomes were modified with anti-CXCR4 antibodies to target metastatic breast cancer (MBC) cells and block migration along the CXCR4-CXCL12 axis. This synergistic approach—coupling the CXCR4 axis blockade with Lcn2 silencing—significantly reduced migration in triple-negative human breast cancer cells (88% for MDA-MB-436 and 92% for MDA-MB-231). The results suggested that drug delivery vehicles engineered to attack multiple migratory pathways may effectively slow progression of MBC.

Abstract P4-01-19: Enhanced sensitivity to detect metastases of breast cancer to the lung and monitor response to inhibition of metastasis by IGF1R inhibitor using SWIFT MRI

Abstract Breast cancers metastasize to the bone, lung, brain and liver and mortality from breast cancer is due to metastases. Drugs that inhibit metastasis in particular of triple negative (TN) breast cancers, more sensitive methods to image metastases and imaging biomarkers to monitor response to these drugs at sites of metastasis are needed to reduce mortality. The type I insulin-like growth factor receptor (IGF1R) regulates metastasis and drugs targeting IGF1R and or insulin receptor inhibit metastasis of TN breast cancer cells in mouse models. The initial clinical trials of these drugs have not been very successful and point to the need to develop imaging biomarkers for this therapy. While MRI is a powerful tool for detecting and imaging cancer, its utility in imaging metastasis to the lung is limited due to the challenges of lung MRI with conventional 3D gradient echo (GRE). MRI does not visualize lung well, mainly due to the abundance of air-tissue interfaces, which cause the MR signal to decay too rapidly for conventional MRI pulse sequences to capture. Clinically lung metastases are monitored by CT or PET but exposure of patients to ionizing radiation is a concern and problematic in longitudinal studies monitoring response to a targeted drug. Further MRI can also be useful in measuring metabolite levels using MR spectroscopy (MRS). Therefore, we evaluated the capability of a novel MR sequence called sweep imaging with Fourier transformation (SWIFT), where the data is acquired concurrently with the radiofrequency pulse, to detect breast cancer metastases to lung and test if inhibition of metastases by an IGF1R targeted drug can be monitored by SWIFT. We used the tail vein injection model of breast cancer metastasis with MDA-MB-231-LM2, a lung-seeking metastatic TN breast cancer cell line. Lung metastases were monitored with bioluminescence imaging (BLI) and MRI every week. MRI was done with two pulse sequences: SWIFT (with a short echo time or TE∼3 μs) and GRE (with a longer TE = 2.2 ms). SWIFT showed significantly higher sensitivity in detecting signals from lung parenchyma compared to GRE and structural information was visible in the area of the lungs. Metastatic tumor growth in the lung induced a progressive increase in signal from the lung parenchyma in SWIFT images. MIP images from SWIFT clearly visualized the lung vascular structures and their disruption due to progression of breast cancer metastasis. To test if SWIFT can also be useful in monitoring inhibition of metastases, mice injected with cells were treated with either vehicle or an IGF1R inhibitor (huEM164) twice a week. When inhibition of metastases in the huEM164 treated group compared to the vehicle group was evident by BLI, MR images were acquired. SWIFT was sensitive in detecting the inhibition of metastases by the IGF1R antibody. Our data show that high sensitivity to fast-decaying signals and tolerance to the magnetic susceptibility in SWIFT enhance the capability to detect signal and structural changes in the lung parenchyma and vasculature due to metastasis of breast cancer. Further, SWIFT was also sensitive to monitor inhibition of metastasis in response to IGF1R targeted drugs. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-01-19.

Metastatic progression of breast cancer: insights from 50 years of autopsies

There remain no clear guidelines for the optimal management of patients with metastatic breast cancer. To better understand its natural history, we undertook a detailed examination of 197 autopsies performed on women who died of breast cancer. We reviewed clinical, treatment and pathological aspects of all cases and, additionally, pathological features and biomarker expression (ER, PgR, HER2, EGFR, p53, Ki67, c-Kit, CK AE1/AE3) were assessed in detail for the primary tumour and matched metastases for 55 of the cases. Genomes of the primary tumour and multiple metastases were analysed by array-based comparative genomic hybridization for six cases##. 945 metastatic deposits were identified, with a median of four/patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%) and non-axillary lymph nodes (55%). Major findings included: (a) patients with CNS metastases were more likely to have bone metastases (p < 0.013); (b) younger age was associated with metastasis to the liver (≤ 49 years; p < 0.001) and to gynaecological organs (≤ 49 years; p = 0.001); (c) surgical excision of the primary tumour was associated with metastasis to the liver (p = 0.002); and (d) ER and PgR showed down-regulation during progression in a non-random manner, particularly in lung/pleura (ER; p < 0.001), liver and bone metastases. Genomic analysis revealed DNA copy number variation between the primary tumour and metastases (e.g. amplification of 2q11.2–q12.1 and 10q22.2–q22.3) but little variation between metastases from the same patient. In summary, the association of CNS and bone metastases, liver and gynaecological metastases in young women and the risk of liver metastases following surgery have important implications for the management of patients with breast cancer. Clonal heterogeneity of the primary tumour is important in developing metastatic propensity and the change in tumour phenotype during progression/colonization highlights the importance of sampling metastatic disease for optimal treatment strategies. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

BRD4 Short Isoform Interacts with RRP1B, SIPA1 and Components of the LINC Complex at the Inner Face of the Nuclear Membrane

Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.

Use of the metastatic breast cancer progression (MBC-P) questionnaire to assess the value of progression-free survival for women with metastatic breast cancer

While overall survival (OS) has historically been the primary endpoint for clinical trials in oncology, progression-free survival (PFS) has gained acceptance as a valuable surrogate endpoint. However, there are no known published reports about the value of PFS from the patient’s perspective. We developed a questionnaire that included items regarding quality of life (QoL) and the importance of different treatment outcomes and presented hypothetical scenarios for which respondents were asked to indicate their preferences concerning treatments as they relate to PFS. 282 women with metastatic breast cancer (MBC), ranging in age from 21 to 80 years completed an online version of this questionnaire. The majority of women (66 %) had been diagnosed with MBC within the previous 3 years and 56 % had been told their MBC had progressed. When asked to rank five treatment characteristics from most important to least important, respondents ranked “extending PFS” as the second most important treatment outcome after OS. When presented with a hypothetical scenario of two women receiving different treatments, respondents preferred the treatment that resulted in longer PFS (16 vs. 12 months), even when OS and side effects were assumed to be equal. Specifically, when asked to consider which woman within the hypothetical scenario had better QoL, physical functioning, and emotional well-being, respondents more often chose the woman who experienced longer PFS (QoL: 40 vs. 6 %; physical functioning: 32 vs. 8 %; emotional well-being: 58 vs. 6 %) compared to the woman within the hypothetical scenario who had a shorter time of progression. Respondents rated their own QoL highest after being told their MBC was responding to treatment (mean score 76.6) versus after the initial diagnosis of breast cancer and MBC (68.5 and 60.3). These findings suggest that extending PFS is an important treatment outcome and, from a patient perspective, improves overall QoL, physical functioning, and emotional well-being.

Identification of epithelial stromal interaction 1 as a novel effector downstream of Krüppel-like factor 8 in breast cancer invasion and metastasis

Krüppel-like factor 8 (KLF8) is a transcriptional factor critical for metastatic progression of breast cancer. Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast-induced gene in non-invasive breast cancer cells is highly overexpressed in invasive breast carcinomas. The function and regulation of EPSTI1, however, remain largely unknown. In this paper, we report a novel KLF8 to EPSTI1 signaling pathway in breast cancer. Using various expression analyses, we revealed a high co-overexpression of KLF8 and EPSTI1 in invasive human breast cancer cells and patient tumors. Ectopic overexpression of KLF8 in the non-invasive, MCF-10A cells induced the EPSTI1 expression, whereas KLF8 knockdown from the invasive, MDA-MB-231 cells decreased the EPSTI1 expression. Promoter activation and binding analyses indicated that KLF8 promoted the EPSTI1 expression by directly acting on the EPSTI1 gene promoter. EPSTI1 knockdown dramatically reduced the KLF8-promoted MCF-10A cell invasion and ectopic expression of EPSTI1 in the non-invasive, MCF-7 cells is sufficient to induce the cell invasion. Experiments using nude mice demonstrated that the ectopic EPSTI1 granted the MCF-7 cells capability of both invasive growth in the breasts and metastasis to the lungs. Using co-immunoprecipitation coupled with mass spectrometry, we discovered that EPSTI1 interacts with the valosin containing protein (VCP), resulting in the degradation of IκBα and subsequent activation of NF-κB in the nucleus. These findings suggest a novel KLF8 to EPSTI1 to VCP to NF-κB signaling mechanism potentially critical for breast cancer invasion and metastasis.

Abstract A52: Loss of p120-catenin induces metastatic progression of breast cancer by inducing anoikis resistance and augmenting growth factor receptor signaling

Abstract The formation of metastasis is the most common cause of breast cancer-related death among women worldwide. A hallmark event in the dissemination of tumor cells from the primary tumor is the loss of cell-cell adhesion. Normally, epithelial integrity is controlled by the adherens junction (AJ), a membrane-tethered structure consisting of E-cadherin and its associated catenins αE-catenin, β-catenin and p120-catenin (p120). Loss of E-cadherin, which results in inactivation of the AJ, is strongly linked to tumor progression. However, little is known about the underlying mechanisms that drive tumor invasion and metastasis. Since p120-catenin (p120) controls E-cadherin turnover at the cell membrane, we investigated the possible tumor suppressor functions of p120 in breast cancer. First, we analyzed a comprehensive set of invasive breast cancers using immunohistochemistry, and observed a lack of p120 protein expression in 33% (97/296). Next, we used a conditional mouse model of noninvasive (p53-driven) mammary carcinoma and introduced a conditional p120 allele. Combined loss of p120 and p53 resulted in tumors that resembled human metaplastic breast cancer and metastasize to lungs and lymph nodes. Knockdown of p120 in in anchorage-dependent breast cancer cell lines induces anoikis resistance by increasing sensitivity to growth factors. Interestingly, we observed secretion of inflammatory cytokines upon knockdown of p120. These cytokines likely underlie the formation of the prometastatic microenvironment in p120 negative mammary carcinomas, which in turn facilitate the production of growth factors. Finally, we performed RNA sequencing on p120-ablated cells and uncovered several highly up regulated growth factor receptors, which we will further examine to determine their eligibility as targets for intervention strategies to better treat p120-negative metastatic breast cancers. Citation Format: Ron C.J. Schackmann, Sjoerd Klarenbeek, Eva J. Vlug, Suzan Stelloo, Miranda van Amersfoort, Milou Tenhagen, Tanya M. Braumuller, Jeroen F. Vermeulen, Petra van der Groep, Ton Peeters, Elsken van der Wall, Paul J. van Diest, Jos Jonkers, Patrick W.B. Derksen. Loss of p120-catenin induces metastatic progression of breast cancer by inducing anoikis resistance and augmenting growth factor receptor signaling. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A52.

Abstract A027: Endothelial-like properties of triple-negative claudin-low breast cancer cells promote tumor-vascular interactions and tumor permeability

Abstract The different intrinsic subtypes of human breast tumors vary in rate and location of metastatic dissemination. In this study, we aimed to determine if tumor-associated vascular properties could help to explain the differences observed in metastagenicity. We used endothelial and breast cancer cell line models to develop gene expression signatures that measured vascular quantity and vascular proliferation. We tested these signatures, amongst other published endothelial signatures, and found that the genomic programs that measured vascular quantity, vascular proliferation, and a VEGF/Hypoxia-signature were the most highly expressed in claudin-low and basal-like tumors. We then tested the vascular signatures on five human breast cancer datasets (n&amp;gt;3,000) and identified two vascular signatures that added prognostic information to intrinsic subtype classification. Next, microvessel density (MVD) scores were obtained from a set of 70 tumors which were also subjected to gene expression profiling. Interestingly, several of the vascular signatures added metastasis-predicting information in addition to MVD (p&amp;lt;0.05). Similar to the tumor data, claudin-low cell lines also expressed high levels of vascular gene signatures as compared to luminal cell lines. Interestingly, pure claudin-low cell lines, and subsets of claudin-low-like cells within established basal-like cell cancer cell lines, exhibited endothelial-like morphology when culture on Matrigel. In vivo xenografts of claudin-low tumors, but not luminal tumors, extensively perfused injected contrast agent through paracellular spaces and non-vascular tumor-lined channels. Taken together, the endothelial-like characteristics of the cancer cells, combined with both the amount and the physiologic state of the vasculature, contribute to breast cancer metastatic progression. We hypothesize that the genetic signatures we have identified highlight patients that should respond most favorably to anti-vascular agents. Citation Format: J. Chuck Harrell, Adam Pfefferle, Nicole Zalles, Aleix Prat, Cheng Fan, Andrey Khramtsov, Olufunmilayo Olopade, Melissa Troester, Andrew Dudley, Charles Perou. Endothelial-like properties of triple-negative claudin-low breast cancer cells promote tumor-vascular interactions and tumor permeability. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A027.

Endothelial-like properties of claudin-low breast cancer cells promote tumor vascular permeability and metastasis

The vasculature serves as the main conduit for breast tumor metastases and is a target of therapeutics in many tumor types. In this study, we aimed to determine if tumor-associated vascular properties could help to explain the differences observed in metastagenicity across the intrinsic subtypes of human breast tumors. Analysis of gene expression signatures from more than 3,000 human breast tumors found that genomic programs that measured vascular quantity, vascular proliferation, and a VEGF/Hypoxia-signature were the most highly expressed in claudin-low and basal-like tumors. The majority of the vascular gene signatures added metastasis-predictive information to immunohistochemistry-defined microvessel density scores and genomically defined-intrinsic subtype classification. Interestingly, pure claudin-low cell lines, and subsets of claudin-low-like cells within established basal-like cancer cell lines, exhibited endothelial/tube-like morphology when cultured on Matrigel. In vivo xenografts found that claudin-low tumors, but not luminal tumors, extensively perfused injected contrast agent through paracellular spaces and non-vascular tumor-lined channels. Taken together, the endothelial-like characteristics of the cancer cells, combined with both the amount and the physiologic state of the vasculature contribute to breast cancer metastatic progression. We hypothesize that the genetic signatures we have identified highlight patients that should respond most favorably to anti-vascular agents.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-013-9607-4) contains supplementary material, which is available to authorized users.

Loss of p120-Catenin Induces Metastatic Progression of Breast Cancer by Inducing Anoikis Resistance and Augmenting Growth Factor Receptor Signaling

Metastatic breast cancer remains the chief cause of cancer-related death among women in the Western world. Although loss of cell-cell adhesion is key to breast cancer progression, little is known about the underlying mechanisms that drive tumor invasion and metastasis. Here, we show that somatic loss of p120-catenin (p120) in a conditional mouse model of noninvasive mammary carcinoma results in formation of stromal-dense tumors that resemble human metaplastic breast cancer and metastasize to lungs and lymph nodes. Loss of p120 in anchorage-dependent breast cancer cell lines strongly promoted anoikis resistance through hypersensitization of growth factor receptor (GFR) signaling. Interestingly, p120 deletion also induced secretion of inflammatory cytokines, a feature that likely underlies the formation of the prometastatic microenvironment in p120-negative mammary carcinomas. Our results establish a preclinical platform to develop tailored intervention regimens that target GFR signals to treat p120-negative metastatic breast cancers.

KLF8 and FAK cooperatively enrich the active MMP14 on the cell surface required for the metastatic progression of breast cancer

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with cancer. The underlying mechanisms, however, remain largely unidentified. We have recently demonstrated that KLF8 expression enhances the activity but not expression of matrix metalloproteinase-2 (MMP2), the target substrate of MMP14. Here, we report a novel KLF8 to MMP14 signaling that promotes human breast cancer invasion and metastasis. Using cell lines for inducible expression and knockdown of KLF8, we demonstrate that KLF8 promotes MMP14 expression at the transcriptional level. Knocking down KLF8 expression inhibited the breast cancer cell invasion both in vitro and in vivo as well as the lung metastasis in mice, which could be rescued by ectopic expression of MMP14. Promoter reporter assays and oligonucleotide and chromatin immunoprecipitations determined that KLF8 activates the human MMP14 gene promoter by both directly acting on the promoter and indirectly via promoting the nuclear translocation of β-catenin, the expression of T cell factor-1 (TCF1) and subsequent activation of the promoter by the β-catenin/TCF1 complex. Inhibition of focal adhesion kinase (FAK) using pharmacological inhibitor, RNA interference or knockout showed that the cell surface presentation of active MMP14 downstream of KLF8 depends upon FAK expression and activity. Taken together, this work identified novel signaling mechanisms by which KLF8 and FAK work together to promote the extracellular activity of MMP14 critical for breast cancer metastasis.

Epithelial to Mesenchymal Transition Promotes Breast Cancer Progression via a Fibronectin-dependent STAT3 Signaling Pathway

We previously established that overexpression of the EGF receptor (EGFR) is sufficient to induce tumor formation by otherwise nontransformed mammary epithelial cells, and that the initiation of epithelial-mesenchymal transition (EMT) is capable of increasing the invasion and metastasis of these cells. Using this breast cancer (BC) model, we find that in addition to EGF, adhesion to fibronectin (FN) activates signal transducer and activator of transcription 3 (STAT3) through EGFR-dependent and -independent mechanisms. Importantly, EMT facilitated a signaling switch from SRC-dependent EGFR:STAT3 signaling in pre-EMT cells to EGFR-independent FN:JAK2:STAT3 signaling in their post-EMT counterparts, thereby sensitizing these cells to JAK2 inhibition. Accordingly, human metastatic BC cells that failed to activate STAT3 downstream of EGFR did display robust STAT3 activity upon adhesion to FN. Furthermore, FN enhanced outgrowth in three-dimensional organotypic cultures via a mechanism that is dependent upon β1 integrin, Janus kinase 2 (JAK2), and STAT3 but not EGFR. Collectively, our data demonstrate that matrix-initiated signaling is sufficient to drive STAT3 activation, a reaction that is facilitated by EMT during BC metastatic progression.

Actigraphy measured sleep disruption as a predictor of survival in advanced breast cancer.

9532 Background: Sleep disruption, prevalent in cancer patients and survivors, is associated with disrupted hormonal circadian rhythms and poor quality of life. Previous studies in cancer patients and survivors have pointed out the association between poor sleep and faster disease progression. However, until now these studies have been limited by their retrospective or correlational design, providing little resolution of the question of whether sleep disruption accelerates disease progression or whether disease progression dysregulates sleep, or whether a third factor might underlie the association between sleep dysregulation and disease progression. This study aimed to clarify this relationship by using a longitudinal research design to examine whether sleep disruption assessed at baseline predicts survival in women with metastatic breast cancer. Methods: We examined sleep quality and duration in 97 women diagnosed with metastatic breast cancer (mean age=54.6, SD=9.8) via wrist-worn actigraphy for 3 days and sleep diaries. Sleep quality was operationalized as poor sleep efficiency (the ratio of total asleep time to total time in bed * 100%). Results: As hypothesized, poor sleep efficiency was found to predict shorter survival (Hazard Ratio (HR), 0.96, 95% CI, 0.93 to 0.98, p&lt;0.001) over 6 years. This relationship remained significant (HR, 0.94, CI, 0.91 to 0.97, p&lt;.001) even after controlling for other known prognostic factors (age, ER status, cancer treatment, metastatic spread, cortisol levels, and depression). Conclusions: Our findings show that sleep dysregulation is a clear and significant independent prognostic factor for disease progression in metastatic breast cancer. Further research is needed to determine whether treating sleep disruption with cognitive behavioral therapy or medication can improve survival in metastatic breast cancer. Funded by P01AG018784, R01CA118867, K07CA132916.

In vivo tibial compression decreases osteolysis and tumor formation in a human metastatic breast cancer model

Bone metastasis, the leading cause of breast cancer-related deaths, is characterized by bone degradation due to increased osteoclastic activity. In contrast, mechanical stimulation in healthy individuals upregulates osteoblastic activity, leading to new bone formation. However, the effect of mechanical loading on the development and progression of metastatic breast cancer in bone remains unclear. Here, we developed a new in vivo model to investigate the role of skeletal mechanical stimuli on the development and osteolytic capability of secondary breast tumors. Specifically, we applied compressive loading to the tibia following intratibial injection of metastatic breast cancer cells (MDA-MB231) into the proximal compartment of female immunocompromised (SCID) mice. In the absence of loading, tibiae developed histologically-detectable tumors with associated osteolysis and excessive degradation of the proximal bone tissue. In contrast, mechanical loading dramatically reduced osteolysis and tumor formation and increased tibial cancellous mass due to trabecular thickening. These loading effects were similar to the baseline response we observed in non-injected SCID mice. In vitro mechanical loading of MDA-MB231 in a pathologically relevant 3D culture model suggested that the observed effects were not due to loading-induced tumor cell death, but rather mediated via decreased expression of genes interfering with bone homeostasis. Collectively, our results suggest that mechanical loading inhibits the growth and osteolytic capability of secondary breast tumors after their homing to the bone, which may inform future treatment of breast cancer patients with advanced disease.

Abstract 2687: Krüppel-like factor 8 targets matrix metalloproteinase-14 to promote breast cancer invasion and metastasis .

Abstract Krüppel-like factor 8 (KLF8) regulates critical gene transcription and invasion events associated with cancer, including epithelial to mesenchymal transition (EMT) and extracellular matrix degradation. Here, we report a novel KLF8-to- matrix metalloproteinase-14 (MMP14) signaling that promotes human breast cancer cell EMT, invasion and metastasis. Cell-surface protein biotinylation and streptavidin pull-down showed that overexpression of KLF8 induced cell-surface accumulation of MMP14 in MCF-10A cells in a FAK-dependent manner, which was correlated with increased MMP14 activity and the ectodomain shedding of E-cadherin. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced expression and activity of MMP14 on the cell surface. Promoter reporter assays and chromatin and oligonucleotide precipitations determined that KLF8 activated the human MMP14 gene promoter both directly and indirectly via beta-catenin. Importantly, Matrigel invasion assays showed that MMP14 was required for the KLF8-induced MCF-10A cell invasion in vitro. Experiments using nude mice demonstrated that MMP-14 overexpression downstream of KLF8 in the MDA-MB-231 cells makes a significant contribution to the invasion and angiogenesis in the local primary tumors and lung metastasis. Taken together, this work identified a novel role and mechanisms for MMP14 downstream of KLF8 in the progression of breast cancer metastasis. Citation Format: Heng Lu, Liu Hu, Lin Yu, Xianhui Wang, Chao Shen, Tianshu Li, Debarati Mukherjee, Satadru Lahiri, Melissa Wason, Jihe Zhao. Krüppel-like factor 8 targets matrix metalloproteinase-14 to promote breast cancer invasion and metastasis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2013-2687