Maja H. Oktay

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.

Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage–Derived VEGFA

UNLABELLED Dissemination of tumor cells is an essential step in metastasis. Direct contact between a macrophage, mammalian-enabled (MENA)-overexpressing tumor cell, and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)] correlates with metastasis in breast cancer patients. Here we show, using intravital high-resolution two-photon microscopy, that transient vascular permeability and tumor cell intravasation occur simultaneously and exclusively at TMEM. The hyperpermeable nature of tumor vasculature is described as spatially and temporally heterogeneous. Using real-time imaging, we observed that vascular permeability is transient, restricted to the TMEM, and required for tumor cell dissemination. VEGFA signaling from TIE2(hi) TMEM macrophages causes local loss of vascular junctions, transient vascular permeability, and tumor cell intravasation, demonstrating a role for the TMEM within the primary mammary tumor. These data provide insight into the mechanism of tumor cell intravasation and vascular permeability in breast cancer, explaining the value of TMEM density as a predictor of distant metastatic recurrence in patients. SIGNIFICANCE Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA signaling from TIE2(hi) TMEM macrophages results in local, transient vascular permeability and tumor cell intravasation. These data provide evidence for the mechanism underlying the association of TMEM with distant metastatic recurrence, offering a rationale for therapies targeting TMEM.

Tumor Microenvironment of Metastasis and Risk of Distant Metastasis of Breast Cancer

BACKGROUND Tumor microenvironment of metastasis (TMEM), consisting of direct contact between a macrophage, an endothelial cell, and a tumor cell, has been associated with metastasis in both rodent mammary tumors and human breast cancer. We prospectively examined the association between TMEM score and risk of distant metastasis and compared risk associated with TMEM score with that associated with IHC4. METHODS We conducted a case-control study nested within a cohort of 3760 patients with invasive ductal breast carcinoma diagnosed between 1980 and 2000 and followed through 2010. Case patients were women who developed a subsequent distant metastasis; control subjects were matched (1:1) on age at and calendar year of primary diagnosis. TMEM was assessed by triple immunostain and IHC4 by standard methods; slides were read by pathologists blinded to outcome. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression, adjusted for clinical variables. A Receiver Operating Characteristic analysis was performed, and the area under the curve was estimated. All statistical tests were two-sided. RESULTS TMEM score was associated with increased risk of distant metastasis in estrogen receptor (ER)(+)/human epidermal growth factor receptor (HER2)(-) tumors (multivariable OR high vs low tertile = 2.70; 95% CI = 1.39 to 5.26; P trend = .004), whereas IHC4 score had a borderline positive association (OR10 unit increase = 1.06; 95% CI = 1.00 to 1.13); the association for TMEM score persisted after adjustment for IHC4 score. The area under the curve for TMEM, adjusted for clinical variables, was 0.78. Neither TMEM score nor IHC4 score was independently associated with metastatic risk overall or in the triple negative or HER2(+) subgroups. CONCLUSIONS TMEM score predicted risk of distant metastasis in ER(+)/HER2(-) breast cancer independently of IHC4 score and classical clinicopathologic features.

Mena invasive (MenaINV) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM

Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena invasive (MenaINV) and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-MenaINV expressing tumor cells are significantly less cohesive and have discontinuous cell–cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more MenaINV mRNA than early non-metastatic ones. Furthermore, MenaINV expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that MenaINV is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that MenaINV expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.

Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast cancer progression

IntroductionAltered expression of Septin 9 (SEPT9), a septin coding for multiple isoform variants, has been observed in several carcinomas, including colorectal, head and neck, ovarian and breast, compared to normal tissues. The mechanisms regulating its expression during tumor initiation and progression in vivo and the oncogenic function of its different isoforms remain elusive.MethodsUsing an integrative approach, we investigated SEPT9 at the genetic, epigenetic, mRNA and protein levels in breast cancer. We analyzed a panel of breast cancer cell lines, human primary tumors and corresponding tumor-free areas, normal breast tissues from reduction mammoplasty patients, as well as primary mammary gland adenocarcinomas derived from the polyoma virus middle T antigen, or PyMT, mouse model. MCF7 clones expressing individual GFP-tagged SEPT9 isoforms were used to determine their respective intracellular distributions and effects on cell migration.ResultsAn overall increase in gene amplification and altered expression of SEPT9 were observed during breast tumorigenesis. We identified an intragenic alternative promoter at which methylation regulates SEPT9_v3 expression. Transfection of specific GFP-SEPT9 isoforms in MCF7 cells indicates that these isoforms exhibit differential localization and affect migration rates. Additionally, the loss of an uncharacterized SEPT9 nucleolar localization is observed during tumorigenesis.ConclusionsIn this study, we found conserved in vivo changes of SEPT9 gene amplification and overexpression during human and mouse breast tumorigenesis. We show that DNA methylation is a prominent mechanism responsible for regulating differential SEPT9 isoform expression and that breast tumor samples exhibit distinctive SEPT9 intracellular localization. Together, these findings support the significance of SEPT9 as a promising tool in breast cancer detection and further emphasize the importance of analyzing and targeting SEPT9 isoform-specific expression and function.

Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration

Analysis of cells in fine-needle aspirate biopsies reveals signals between breast cancer cells and macrophages that promote metastasis. Retaining a Barrier to Metastasis Metastatic disease results from the migration of cancer cells out of the primary tumor and invasion into neighboring tissue or vasculature and establishment of secondary sites. Using an in vitro transendothelial migration assay and cells obtained from fine-needle aspiration (FNA) biopsies from patients with invasive breast cancer, Pignatelli et al. examined the molecular and cellular interactions that may enable the migration of cancer cells into blood vessels (see the Perspective by Kiersse et al.). Where cancer cells colocalized with macrophages in the assay, contacts between endothelial cells were degraded. Macrophages promoted cancer cell migration by secreting the growth factors EGF and CSF-1, and cancer cells in turn secreted CSF-1, which functioned as a paracrine signal to macrophages and for some cancer cell subtypes also as an autocrine signal. Blocking the interaction of CSF-1 with its receptor prevented the transendothelial migration of cancer cells in culture, suggesting that this may be one approach to prevent metastatic progression in cancer patients. Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)–responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding MenaINV, an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down MenaINV or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that MenaINV and TMEM frequency are correlated prognostic markers and CSF-1 and MenaINV may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism

Chemotherapy induces prometastatic changes in breast cancer, reversible by TIE2 or MENA inhibition. Closing the door to cancer cells Breast cancer is one of the most common tumor types, and metastasis greatly increases the risk of death from this disease. By studying the process of intravasation or entry of cells into the vasculature, Karagiannis et al. discovered that, in addition to killing tumor cells, chemotherapy treatment can also increase intravasation. Groups of cells collectively known as tumor microenvironment of metastasis (TMEM) can serve as gateways for tumor cells entering the vasculature, and the authors discovered that several types of chemotherapy can increase the amounts of TMEM complexes and circulating tumor cells in the bloodstream. The researchers also determined that a drug called rebastinib can interfere with TMEM activity and help overcome the increased risk of cancer cell dissemination. Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

Autocrine CSF1R signaling mediates switching between invasion and proliferation downstream of TGFβ in claudin-low breast tumor cells

Patient data suggest that colony-stimulating factor-1 (CSF1) and its receptor (CSF1R) have critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cell lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cell lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight-junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ.

Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena INV -initiated invadopodium formation

The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/MenaINV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell – macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.

Septin 9 amplification and isoform-specific expression in peritumoral and tumor breast tissue

Abstract Septins are a large family of GTP-binding proteins abnormally expressed in many solid tumors. Septin 9 (SEPT9) in particular has been found overexpressed in diverse human tumors including breast, head and neck, ovarian, endometrial, kidney, and pancreatic cancer. Although we previously reported SEPT9 amplification in breast cancer, we now show specifically that high-grade breast carcinomas, the subtype with worst clinical outcome, exhibit a significant increase in SEPT9 copy number when compared with other tumor grades. We also present, for the first time, a sensitive and quantitative measure of seven (SEPT9_v1 through SEPT9_v7) isoform variant mRNA levels in mammary epithelial cells. SEPT9_v1, SEPT9_v3, SEPT9_v6, and SEPT9_v7 isoforms were expressed at the highest levels followed by SEPT9_v2 and SEPT9_v5, whereas SEPT9_v4 was almost undetectable. Although most of the isoforms were upregulated in primary tumor tissues relative to the patient-matched peritumoral tissues, SEPT9_v4 remained the lowest expressing isoform. This comprehensive analysis of SEPT9 provides substantial evidence for increased SEPT9 expression as a consequence of genomic amplification and is the first study to profile SEPT9_v1 through SEPT9_v7 isoform-specific mRNA expression in tumor and nontumor tissues from patients with breast cancer.