Search

Significance In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities., Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.

The majority of breast cancer patient deaths occur when tumor cells migrate away from the primary tumors and disseminate metastatically. Cancer cells at the invasive front mimic the behaviors of non-tumorigenic epithelial cells at wound edges but show less coordinated migration comparatively. While most wound healing studies use a timeframe of 24-48 hours, our previous studies have shown that ATP released from wounded cells initiates a calcium (Ca2+) signal within seconds that spreads to neighboring cells through activation of the P2Y2 surface receptor. RNAseq data of MCF10A mammary epithelial cells with stepwise mutations of PTEN knockout and KRas activation alone or in combination, demonstrate that P2Y2 is downregulated specifically by active KRas. Fluorescence microscopy was used to measure Ca2+ signaling and mitochondrial membrane potential in parental and mutant MCF10A cells. Change in total fluorescence was measured and compared to baseline using Fluo-4, a Ca2+ fluorescent indicator, and TMRM (tetramethylrhodamine, methyl ester), a fluorescent mitochondrial membrane potential dye on Nikon analysis software. We show that KRas activation disrupts ATP stimulation and Ca2+ signaling. This change was further quantified using a FlexStation III plate reader with Fluo-4 to read specific fluorescent changes in Ca2+ after ATP addition. ATP-stimulated Ca2+ was also disrupted in other breast tumor cell lines harboring Ras activating mutations, MDA-MB-231 and MDA-MB-436, both showing a similar decrease in P2Y2 expression. These data show that ATP stimulation can be used to further understand the differences in Ca2+ signaling and mechanical-response between normal breast epithelial cells and cancer cells. Clarifying these molecular mechanisms could reveal targets for new cancer treatments, especially since 90% of human tumors are epithelial carcinomas. Citation Format: Makenzy Mull, Stephen Pratt, Rachel Lee, Abanoub Gad, Katarina Chang, David Annis, Keyata Thompson, Michele Vitolo, Liron Boyman, Stuart Martin. Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2425.

Melanoma cell adhesion molecule (MCAM) is a protein located on the cell surface that was first discovered in advanced primary tumors and metastatic lesions. Overexpression of MCAM is associated with triple-negative breast cancer, decreased patient survival, and induces vimentin and slug indicative of an epithelial to mesenchymal transition (EMT). Additionally, breast cancer cell lines overexpressing MCAM show an increase in migration, invasion, and tumorigenesis. Given that overexpressed MCAM causes increased migration and invasion in multiple cancer types, MCAM could be one of the major contributors to recent studies showing increased metastatic phenotypes with certain cancer treatments. Decitabine (5-Aza-dC) is a chemotherapeutic used for acute myeloid leukemias with clinical trials underway for breast cancer use. 5-Aza-dC results in the depletion of DNA methyltransferase and acts as a demethylating agent. In 2019, 5-Aza-dC was reported to increase mRNA and protein levels of MCAM in various breast cancer cell lines, followed by an increase in EMT markers. Studies suggest that 5-Aza-dC treatment in chemo-sensitive ovarian cancer cell lines results in increased EMT markers as well as increased invasion and migration. Microtubules are a very common target of multiple cancer therapies. However, the impact of 5-Aza-dC and MCAM on the microtubule network and non-adherent cells has not been well studied. We set out to explore changes in microtentacle (McTN), cell attachment and clustering, and microtubule modifications due to 5-Aza-dC treatment and MCAM expression. Treatment of MCF10A, MCF10A variants, and MCF7 breast cancer cell lines with 5-Aza-dC showed consistent increases in the expression of MCAM. When exploring the effects of 5-Aza-dC treatment on the cytoskeleton, specifically microtubules, it was found that there is a significant increase in acetyl tubulin and detyrosinated tubulin, two common microtubule modifications correlated with an increase of McTNs. However, there was not a significant increase in the number of cells with McTNs in treated cells. Since McTNs can aid in attachment and cell clustering, attachment assays and clustering assays were performed after 5-Aza-dC treatment. These experiments showed that 5-Aza-dC decreased the ability of all cell lines to attach and impeded these cells' ability to form clusters. While there are emerging concerns that Decitabine can increase metastatic phenotypes of migration and invasion in adherent cells, our results indicate that within a non-adherent environment, Decitabine reduces the metastatic phenotypes of clustering and reattachment. Ongoing experiments focus on testing the impact of stable genetic alterations in these cell lines to examine the specific impact of MCAM overexpression on the microtubule network and metastatic potential. Citation Format: David Annis, Keyata Thompson, Julia Ju, Makenzy Mull, Trevor Mathias, Michele Vitolo, Stuart Martin. 5-Aza-dC treatment impacts the microtubule network to decrease metastatic potential through reduced cell clustering and attachment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 965.

Background: Clinical breast cancer imaging inevitably focuses on tumor growth rather than the metastatic dissemination that is a greater challenge for patient survival. Emerging preclinical evidence indicates that chemotherapy can elevate levels of circulating tumor cells (CTCs) and increase metastatic recurrence. Targeting metastatic phenotypes of CTCs could reveal therapeutic strategies to reduce metastasis that would be overlooked by measurements of tumor growth.Methods: We investigated how the FDA-approved microtubule-depolymerizing Vinca alkaloid, Vinorelbine, affects three metastatic phenotypes of reattachment, clustering and microtentacles (McTNs). Microfluidic cell tethering technology (TetherChip) was used to measure Vinorelbine effects on McTNs and clustering while xCelligence impedance was used for reattachment assays. Bioluminescence imaging monitored tumor growth and metastasis in mice. ANGLE Parsortix and Vortex Biosciences VTX-1 were used to capture live tumor cells from blood samples for confocal microscopy to rapidly measure tumor cell responses to Vinorelbine.Results: We demonstrate that a focused (1h) Vinorelbine treatment is sufficient to inhibit reattachment, clustering and McTNs in non-adherent breast tumor cells. Quantitative analysis of treated non-adherent cells reveals that McTNs are significantly lower in number (p= 0.012) and shorter in length (p= 0.000034). Treating mice with Vinorelbine (5mg/kg) for only 24h did not significantly affect primary tumor survival. However, median metastatic tumor survival after injection of circulating tumor cells extended from 8 weeks to 30 weeks after a focused 24h treatment with Vinorelbine. Microtentacle inhibition by Vinorelbine was also detectable within 1h, using live tumor cells isolated from blood samples and analyzed with confocal microscopy for McTNs on TetherChip microfluidic surfaces. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies.Conclusions: This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth. Moreover, the anti-metastatic actions of Vinorelbine could be detected in less than one hour using microfluidic TetherChip technology, establishing a new approach for precision medicine aimed at reducing metastasis.

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 μm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

Summary The mammosphere assay has become widely employed to quantify stem-like cells in a population. However, the problem is there is no standard protocol employed by the field. Cell seeding densities of 1,000 to 100,000 cells/mL have been reported. These high densities lead to cellular aggregation. To address this, we have individually tracked 1,127 single MCF-7 and 696 single T47D human breast tumor cells by eye over the course of 14 days. This tracking has given us detailed information for the commonly used endpoints of 5, 7, and 14 days that is unclouded by cellular aggregation. This includes mean sphere sizes, sphere-forming efficiencies, and a well-defined minimum size for both lines. Importantly, we have correlated early cell division with eventual sphere formation. At 24 hr post seeding, we can predict the total spheres on day 14 with 98% accuracy in both lines. This approach removes cell aggregation and potentially shortens a 5- to 14-day assay to a 24 hours., Graphical Abstract, Highlights • Single-cell tracking removes confounding aggregation from the mammosphere assay • Tracking reveals sphere-forming efficiencies much higher than commonly reported • True clonal spheres are smaller than commonly reported • At 24 hours, tracking can predict total day 14 spheres with 98% accuracy, Biology Experimental Methods; Cancer

Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500μm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

The challenge of predicting which patients with breast cancer will develop metastases leads to the overtreatment of patients with benign disease and to the inadequate treatment of the aggressive cancers. Here, we report the development and testing of a microfluidic assay that quantifies the abundance and proliferative index of migratory cells in breast-cancer specimens, for the assessment of their metastatic propensity and for the rapid screening of potential antimetastatic therapeutics. On the basis of the key roles of cell motility and proliferation in cancer metastasis, the device accurately predicts the metastatic potential of breast-cancer cell lines and of patient-derived xenografts. Compared to unsorted cancer cells, highly motile cells isolated by the device exhibited similar tumourigenic potential but markedly increased metastatic propensity in vivo. RNA sequencing of the highly motile cells revealed an enrichment of motility-related and survival-related genes. The approach might be developed into a companion assay for the prediction of metastasis in patients and for the selection of effective therapeutic regimens.

A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common.

The presence of tumor cells in the circulation is associated with a higher risk of metastasis in patients with breast cancer. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. AMP-activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We report that AMPK can regulate the cytoskeleton of breast cancer cells in both attached and suspended conditions. We tested the effects of AMPK on microtubule stability and the actin-severing protein, cofilin. AMPK inhibition with compound c increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation with A-769662 decreased microtubule stability and cofilin activation with concurrent decreases in McTN formation and cell re-attachment. This data shows for the first time that AMPK shifts the balance of cytoskeletal forces in suspended breast cancer cells, which affect their ability to form McTNs and re-attach. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of breast tumor cells.

// Lekhana Bhandary 1,2 , Rebecca A. Whipple 1 , Michele I. Vitolo 1,2,3 , Monica S. Charpentier 1 , Amanda E. Boggs 1 , Kristi R. Chakrabarti 1,2 , Keyata N. Thompson 1 and Stuart S. Martin 1,2,3 1 University of Maryland School of Medicine, Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, University of Maryland, School of Medicine, Baltimore, Maryland, USA 2 Graduate Program in Molecular Medicine, University of Maryland, School of Medicine, Baltimore, Maryland, USA 3 Department of Physiology, University of Maryland, School of Medicine, Baltimore, Maryland, USA Correspondence: Stuart S. Martin, email: // Keywords : ROCK, microtentacles, Y-27632, breast cancer metastasis, reattachment Received : November 24, 2014 Accepted : January 12, 2015 Published : January 31, 2015 Abstract The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles , can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.

Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1–mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator–activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a β-galactoside–binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galβ1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD 100 ) that binds gal3 with picomolar affinity. TFD 100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD 100 . Because the gal3–TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD 100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.

During metastasis, invading cells produce various actin-based membrane protrusions that promote directional migration and proteolysis of extracellular matrix (ECM). Observations of actin staining within thin, tubulin-based microtentacle (McTN) protrusions in suspended MDA-MB-231 tumor cells prompted an investigation of whether McTNs are structural or functional analogs of invadopodia. We show here that MDA-MB-231 cells are capable of producing invadopodia and McTNs, both of which contain F-actin. Invadopodium formation was enhanced by expression of a constitutively-active c-Src kinase, and repressed by expression of dominant negative, catalytically-inactive form of c-Src. In contrast, expression of inactive c-Src significantly increased McTN formation. Direct inhibition of c-Src with the SU6656 inhibitor compound also significantly enhanced McTN formation, but suppressed invadopodia, including the appearance of F-actin cores and phospho-cortactin foci, as well as completely blocking focal degradation of extracellular matrix. Additionally, silencing of Tks5 in Src-transformed fibroblasts blocked invadopodia without affecting McTNs. Genetic modification of c-Src activity that promoted McTN formation augmented capillary retention of circulating tumor cells in vivo and rapid re-attachment of suspended cells in vitro, even though invadopodia were strongly suppressed. These results indicate that McTNs are capable of enhancing tumor cell reattachment, even in the absence of Tks5 and active Src, and define separate cytoskeletal mechanisms and functions for McTNs and invadopodia.

Background The heart develops under reduced and varying oxygen concentrations, yet there is little understanding of oxygen metabolism in the normal and mal‐development of the heart. Here we used a novel reagent, the ODD ‐Luc hypoxia reporter mouse ( o xygen d egradation d omain, ODD ) of Hif‐1 α fused to Luciferase (Luc), to assay the activity of the oxygen sensor, prolyl hydroxylase, and oxygen reserve, in the developing heart. We tested the role of hypoxia‐dependent responses in heart development by targeted inactivation of Hif‐1 α. Methods and Results ODD ‐Luciferase activity was 14‐fold higher in mouse embryonic day 10.5 (E10.5) versus adult heart and liver tissue lysates. ODD ‐Luc activity decreased in 2 stages, the first corresponding with the formation of a functional cardiovascular system for oxygen delivery at E15.5, and the second after birth consistent with complete oxygenation of the blood and tissues. Reduction of maternal inspired oxygen to 8% for 4 hours caused minimal induction of luciferase activity in the maternal tissues but robust induction in the embryonic tissues in proportion to the basal activity, indicating a lack of oxygen reserve, and corresponding induction of a hypoxia‐dependent gene program. Bioluminescent imaging of intact embryos demonstrated highest activity in the outflow portion of the E13.5 heart. Hif‐1 α inactivation or prolonged hypoxia caused outflow and septation defects only when targeted to this specific developmental window. Conclusions Low oxygen concentrations and lack of oxygen reserve during a critical phase of heart organogenesis may provide a basis for vulnerability to the development of common septation and conotruncal heart defects.

Cancer stem–like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non–stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell–cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy. Cancer Res; 74(4); 1250–60. ©2013 AACR.

Introduction Detyrosinated tubulin, a post-translational modification of α-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilitate tumor cell reattachment to endothelial layers. Given the induction of EMT associated with inflammation and cancer progression, we tested anti-inflammatory nuclear factor-kappaB (NF-κB) inhibitors on a panel of human breast carcinoma cells to examine their effects on detyrosinated tubulin to identify more specific tubulin-directed anti-cancer treatments. Methods Using metastatic human breast carcinoma cells MDA-MB-157, MDA-MB-436, and Bt-549, we measured the impact of NF-κB inhibitors parthenolide, costunolide, and resveratrol on detyrosinated tubulin using protein expression analysis and immunofluorescence. A luciferase reporter assay and a viability screen were performed to determine if the effects were associated with their NF-κB inhibitory properties or were a result of apoptosis. Real-time monitoring of cell-substratum attachment was measured utilizing electrical impedance across microelectronic sensor arrays. We compared the selectivity of the NF-κB inhibitors to specifically target detyrosinated tubulin with traditional tubulin-targeted therapeutics, paclitaxel and colchicine, throughout the study. Results Sesquiterpene lactones, parthenolide and costunolide, selectively decrease detyrosinated tubulin independent of their inhibition of NF-κB. Live-cell scoring of suspended cells treated with parthenolide and costunolide show reduction in the frequency of microtentacles and inhibition of reattachment. Structural analysis shows that parthenolide and costunolide can decrease detyrosinated microtubules without significantly disrupting the overall microtubule network or cell viability. Paclitaxel and colchicine display indiscriminate disruption of the microtubule network. Conclusions Our data demonstrate that selective targeting of detyrosinated tubulin with parthenolide and costunolide can reduce McTN frequency and inhibit tumor cell reattachment. These actions are independent of their effects on NF-κB inhibition presenting a novel anti-cancer property and therapeutic opportunity to selectively target a stable subset of microtubules in circulating tumor cells to reduce metastatic potential with less toxicity in breast cancer patients.

Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN(-/-) cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN(-/-) cells. One such actin-severing protein, cofilin, is activated in detached PTEN(-/-) cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN(-/-) cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN(-/-) cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN(-/-) and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology.

Breast cancer is the most common nonskin cancer and is the second leading cause of cancer-related deaths in women. Most methods of intervention involve combinations of surgery, chemotherapy, and ionizing radiation. Both chemotherapy and ionizing radiation can be effective against many types of cancer, but they also harm normal tissues. The use of nonionizing, magnetic fields has shown early promise in a number of in vitro and animal studies. Our study tested the effect of varying durations of magnetic exposure on tumor growth and viability in mice injected with breast cancer cells. Cancer cells were labeled through stable expression of firefly luciferase for monitoring of tumor growth and progression by using an in vivo imaging system. We hypothesized that magnetic field exposure would influence tumor growth and progression. Our results showed that exposure of the mice to magnetic fields for 360 min daily for as long as 4 wk suppressed tumor growth. Our study is unique in that it uses an in vivo imaging system to monitor the growth and progression of tumors in real time in individual mice. Our findings support further exploration of the potential of magnetic fields in cancer therapeutics, either as adjunct or primary therapy.

The cytoskeletal organization of detached and circulating tumor cells is currently not well-defined and may provide potential targets for new therapies to limit metastatic tumor spread. In vivo, circulating tumor cells reattach in distant tissues via a mechanism that is tubulin-dependent and suppressed by polymerized actin. The cytoskeletal mechanisms that promote reattachment of circulating tumor cells match exactly with the mechanisms supporting tubulin microtentacles, which we have recently identified in detached breast tumor cells. In this study, we aimed to investigate how microtentacle formation is affected by the microtubule-associated protein, tau, which is expressed in a subset of chemotherapy-resistant breast cancers. We demonstrate that endogenous tau protein localizes to microtentacles and is both necessary and sufficient to promote microtentacle extension in detached breast tumor cells. Tau-induced microtentacles increase reattachment of suspended cells and retention of circulating tumor cells in lung capillaries. Analysis of patient-matched primary and metastatic tumors reveals that 52% possess tau expression in metastases and 26% display significantly increased tau expression over disease progression. Tau enrichment in metastatic tumors and the ability of tau to promote tumor cell reattachment through microtentacle formation support a model in which tau-induced microtubule stabilization provides a selective advantage during tumor metastasis.

Breast cancer is the most frequent malignancy in women, and although many breast cancers are curable via surgery, approximately one quarter maintain a latent and insidious characteristic of slow growth. The loss of the tumor suppressor PTEN is associated with breast cancer stage, increased lymph node status, and disease-related death, and the high rate of loss in primary tumors suggests a potential role in initiation and/or progression of the disease. Additionally, a large fraction of breast tumors carry oncogenic mutations resulting in the hyper-activation of the MAPK/ERK cascade (20%-25% ErbB2, 5% KRAS, 2% BRAF, 1% HRAS, 1% NRAS). Hyperactivation of survival and growth pathways is considered a hallmark of many human carcinomas, including breast cancer. The overactivation of the PI3K pathway (PTEN loss) or the MAPK pathway could grant a cell the ability to circumvent inhibitory pathways. However, specific cellular alterations in human breast epithelium controlled by PTEN inactivation and/or Ras activation, which lead to early primary tumor formation, remain poorly defined. Since the current view of cancer is based on a “multi-hit” hypothesis where human cancers display a multitude of genetic and epigenetic changes, and a number of such alterations are required for tumor development, the loss of PTEN (activation of the PI3K pathway) and expression of activated K-Ras(V12) (activation of the MAPK pathway) may cooperate to promote tumorigenesis. We therefore tested the hypothesis that the activation of the MAPK pathway via activated Ras expression in a PTEN-negative background promotes tumorgenicity. Using the non-tumorigenic human mammary cells line, MCF-10A, we created MCF-10A PTEN-/- cells, MCF-10A KRas(V12) cells, and MCF-10A PTEN-/-KRas(V12) cells. We have found that each mutation, independently and collectively, greatly enhanced cellular survival and regrowth efficiency of nutrient deprived and suspended cells in vitro. Using bioluminescent mouse xenograft models, we have determined the cells with either PTEN loss or KRas(V12) expression maintain an increased persistence in vivo up to 5 weeks beyond that of the parental cells, and the combination of PTEN loss and KRas(V12) expression resulted large tumors within 4 weeks of initial injection. The combination of “one-hit” to the PI3K pathway and “one-hit” to the MAPK pathways synergized to result in aggressive tumor growth, while each individual mutation only lead to cellular persistence in vivo, a characteristic that may have been previously overlooked in less sensitive xenograft models, either without bioluminescence imaging or when transplanting less genetically stable tumor cells. Citation Format: Keyata N. Thompson, Rebecca A. Whipple, Jennifer R. Yoon, Monica S. Charpentier, Amanda E. Boggs, Lekhana Bhandary, Kristi R. Chakrabarti, Stuart S. Martin, Michele I. Vitolo. Activation of the MAPK pathway in combination with PTEN loss leads to aggressive primary tumor formation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2161. doi:10.1158/1538-7445.AM2015-2161

Breast cancer is the most frequent malignancy in women, and although many breast cancers are curable via surgery, approximately one quarter maintain a latent and insidious characteristic of slow growth with early metastasis. The loss of the tumor suppressor PTEN is associated with breast cancer stage, increased lymph node status, and disease-related death, and the high rate of loss in primary tumors suggests a potential role in initiation and/or progression of the disease. However, specific cellular alterations in human breast epithelium controlled by PTEN inactivation, which lead to an increased metastatic phenotype, remain poorly defined. Many breast cancers have an activated PI3K pathway either due to PTEN loss or PI3K mutation, while a large fraction of breast tumors carry oncogenic mutations that cause hyperactivation of the MAPK/ERK cascade (20%–25% ErbB2, 5% KRAS, 2% BRAF, 1% HRAS, 1% NRAS). Using the isogenic non-tumorigenic MCF-10A and MCF-10A PTEN-/- cells, we have determined that PTEN-/- cells persist in mouse xenografts while their isogenic counterparts disappear. Although the PTEN-/- cells form small, 2mm tumors, these cells do not produce large growths. Since the current view of cancer is based on a “multi-hit” hypothesis: human cancers display a multitude of genetic and epigenetic changes, and a number of such alterations are required for the step-wise progression of tumor development, the activation of both signaling pathways may cooperate to promote tumorigenesis. We therefore tested the hypothesis that the activation of the MAPK pathway in a PTEN-negative background promotes tumorgenicity. To determine the cooperativity of PTEN loss and the MAPK pathway, we have expressed activated KRAS(V12) in the MCF-10A PTEN-/- cells. The combination of PTEN loss and KRas(V12) expression resulted large tumors within 4 weeks of injection. Interestingly, we discovered that “one-hit” of either PTEN loss or active Ras leads to a cellular persistence in vivo, a characteristic that may have been previously overlooked in more genetically unstable tumor cells. Citation Format: Keyata N. Thompson, Rebecca A. Whipple, Monica S. Charpentier, Amanda E. Boggs, Lekhana Bhandray, Kristi R. Chakrabarti, Stuart S. Martin, Michele I. Vitolo. Cell dormancy and tumorigenicity due to PTEN loss. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A32.

Circulating Tumor Cells (CTCs) predict poor patient outcome and increased CTC detection is correlated with higher risk of metastasis. The efficiency of cancers to metastasize depends on the survival and reattachment of CTCs in distant tissues. Our lab has demonstrated the significance of microtubule based cellular extensions, termed microtentacles, in enhancing CTC aggregation and reattachment to the endothelial wall. Microtentacles (McTNs) are highly dynamic protrusions formed in free-floating suspended cells and occur at higher frequencies in more metastatic cell lines. Recently, there has been interest generated in using ROCK as a therapeutic target in cancers based on emerging data that over-activation of Rho/ROCK pathway plays a role in tumor development and metastasis. ROCK inhibitors are being considered for clinical trials because of their pre-clinical success in reducing tumor cell migration, invasion and metastasis. However, the effect of Rho/ROCK inhibition on CTC reattachment and metastasis has not been well established. ROCK promotes cellular contraction by phosphorylating non-muscle myosin II (NMII), allowing it to crosslink actin, as well as by phosphorylating and inhibiting the activity of Myosin Light Chain Phosphatase (MLCP). In a normal, noncancerous cell, the outward expansion of microtubules is balanced by the inward contraction of the actin cortex. ROCK inhibition decreases cell contractility and shifts the balance towards a looser, more relaxed actin cortex that is unable to contain the outward growth of microtubules. Western blot analysis of attached and suspended cells treated with a ROCK inhibitor showed a decrease in phosphorylated MLCP and NMII in the breast cancer cell lines, BT549 and Hs578T. Treatment with ROCK inhibitor significantly increased the number of cells expressing McTNs. The increase in McTNs corresponded with increased in vitro cell reattachment, measured by the xCelligence, Real-Time Cell Analyser. Knocking down ROCK1 and ROCK2 with silencing RNAs also resulted in increased McTN counts. Conversely, increasing cell contractility by treating cells with a Rho pathway activator led to a reduction in McTNs and cell attachment. These results show that ROCK inhibitors could actually promote metastasis by increasing attachment of CTCs to the vasculature. A better understanding of the effect of cancer therapies on all stages of metastasis will prevent the use of drugs that could reduce tumor growth and motility in the short-term but result in increasing circulating tumor cells and elevating metastatic potential in the long-term. Citation Format: Lekhana Bhandary, Michele I. Vitolo, Rebecca A. Bettes, Monica S. Charpentier, Amanda E. Boggs, Jana Slovic, Keyata Thompson, Stuart S. Martin. ROCK inhibition promotes microtentacle formation and reattachment of breast cancer cells. [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 3160. doi:10.1158/1538-7445.AM2014-3160

Circulating tumor cells (CTCs) have been identified as a predictive factor of patient outcome and disease progression in breast cancer. Solid tumor metastasis involves detachment of epithelial cells into the lymphatics or vasculature, and the subsequent reattachment in distant tissues to establish new tumors at metastatic sites. Our lab has identified tubulin-based protrusions, termed microtentacles (McTNs), which facilitate tumor cell re-attachment and can penetrate endothelial layers to promote the retention of CTCs in distant tissues. In the current study, we examine the role of the physiological cytokine, TGFβ, in the cytoskeletal changes that promote McTN formation. McTNs are induced during the epithelial-to-mesenchymal transition (EMT) that accompanies tumor progression, and previous studies have shown that TGFβ can promote an EMT in a pathologic setting, despite its normal role as an anti-proliferative factor. When we treated MCF10A nontumorigenic mammary epithelial cells and MCF-7 breast tumor cells with TGFβ, immunoblotting demonstrated changes in protein expression consistent with an EMT, such as increased vimentin and N-Cadherin and decreased E-Cadherin expression. Alongside these typical EMT-induced protein expression changes, levels of detyrosinated α-tubulin (Glu-tubulin) also increased, which is a marker of stabilized microtubules that also promotes McTN formation. Furthermore, immunofluorescence demonstrated that Glu-tubulin was highly diffuse in untreated cells, but became organized into distinct cytoplasmic filaments upon treatment of cells with TGFβ. Detached MCF10A and MCF-7 cells also showed increased McTNs following exposure to TGFβ. Real-time measurement of cell reattachment by electrical impedance demonstrated that the increased McTNs associated with TGFβ treatment resulted in more efficient and faster cell reattachment. These results emphasize that the post-translational alterations in microtubules promoted by TGFβ can promote increased McTNs and that therapies targeting the molecular mechanisms underlying these TGFβ-induced cytoskeletal changes could provide new targets aimed at reducing CTC reattachment and metastasis. Citation Format: Jana Slovic, Michele I. Vitolo, Rebbecca A. Whipple, Amanda E. Boggs, Monica S. Charpentier, Lekhana Bhandary, Keyata Thompson, Stuart S. Martin. Microtentacle stabilization during TGFβ-induced EMT as a new therapeutic target to reduce circulating tumor cell 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 276. doi:10.1158/1538-7445.AM2013-276

Breast cancer is the 2nd leading cause of cancer-related death among women; however, the majority of deaths arise as complications from metastasis, rather than the primary tumor. The cancer stem cell (CSC) hypothesis provides an explanation for the limited success of current therapies for metastatic breast cancer. CSCs are defined as a subpopulation of tumor-initiating cells with the stem cell-like characteristics of self-renewal and multipotency. CSCs have been shown to enter circulation and reach distal tissues, where they may remain cell-cycle arrested, and therefore resistant to conventional chemotherapeutics. Recent work in our lab has shown that circulating tumor cells use dynamic tubulin-based microtentacles (McTNs) to reattach to distant tissues, a critical step in metastasis. McTNs are novel cellular structures formed by epithelial cells when detached from the extracellular matrix and are increased in more metastatic breast cancer cell lines. Given the proposed metastatic efficiency of CSCs, we examined McTN incidence and function in mammary stem cells and breast cancer stem cells. Flow cytometry for the CSC markers CD44 and CD24 showed that breast tumor cell lines with increased CSC characteristics display higher McTN frequencies. Given this correlation, CD44 and CD24 immunofluorescence was used to separate human mammary epithelial (HMLE) cells into CSC and non-CSC subpopulations with flow cytometry. Stem-like HMLE cells (CD44hi/CD24lo) have increased McTN levels compared to non-CSC cells (CD44lo/CD24hi) from the same HMLE cell line. The CSC subpopulation also demonstrated increased cytoskeletal modifications that promote McTN formation, such as elevated vimentin expression and an increase in both the amount and the bundling of stabilized, detyrosinated tubulin. Vimentin and detyrosinated tubulin localize to McTNs in suspended stem-like HMLEs. The increased McTNs in stem-like HMLEs promote faster initial reattachment of suspended cells that is inhibited by the tubulin-directed drug, Colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live cell confocal microscopy demonstrates that McTNs participate in the structure of breast stem cell mammopsheres, extending between adjacent cells along cell-cell junctions. McTNs can be reduced by the breast CSC targeting agent Curcumin. These studies show that McTNs contribute to the metastatic potential of breast CSCs as well as the ability of mammary stem cells to form multicellular mammospheres. We anticipate that this work will clarify the molecular mechanisms underlying tubulin alterations in breast cancer stem cells and identify how these tubulin modifications may be targeted more specifically to reduce McTNs and the metastatic reattachment efficiency of breast cancer CSCs. Citation Format: Monica S. Charpentier, Rebecca A. Bettes, Michele I. Vitolo, Amanda E. Boggs, Jana Slovic, Keyata Thompson, Lekhana Bhandary, Jennifer Yoon, Stuart S. Martin. The role of microtentacles in the metastatic potential of breast tumor stem cells. [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 3772. doi:10.1158/1538-7445.AM2013-3772

Breast cancer is the most frequent malignancy in women, and although many breast cancers are curable via surgery, approximately one quarter maintain a latent and insidious characteristic of slow growth with early metastasis. The loss of the tumor suppressor PTEN has been associated with breast cancer stage, lymph node status, and disease-related death, and the high rate of loss in primary tumors suggests a potential role in initiation and/or progression of the disease. However, specific cellular alterations in human breast epithelium controlled by PTEN inactivation, which lead to an increased metastatic phenotype, remain poorly defined. We have recently determined that PTEN expression loss leads to the production of long, dynamic, tubulin-based membrane protrusions upon detachment, which increase in frequency, number and length per cell compared to their isogenic, PTEN-expressing parental counterparts. These novel structures, termed microtentacles (McTNs), are structurally distinct from classical actin-based extensions of adherent cells, persist for days in breast tumor lines that are resistant to anoikis, and aid in the reattachment to matrix or cell monolayers and homo- and heterotypic aggregation. McTNs form when the balance of cytoskeletal forces shifts. In order to control morphology, normal cells counteract the expansion of microtubules with tension from the actin cortex. However, altering the balance between microtubules and actin has serious implications for circulating tumor cells (CTCs) dissemination, as metastatically efficient CTCs have been observed to avoid shear-induced fragmentation by undergoing sphere-to-cylinder shape transformations within capillaries. We, therefore, tested the hypothesis that PTEN loss disrupts the actin cortex to allow the increased production of McTNs. We determined that suspended PTEN-null mammary epithelial cells maintained elevated activation of the PI3K/Akt and MAPK pathways and apoptotic resistance to cell rounding and matrix detatchment, but neither activated pathway was responsible for the increased McTNs in these cells. The McTNs produced in the PTEN-null cells aid in cell reattachment, spreading, and homotypic aggregation, and Western blot analysis has proven that the PTEN-null cells show reduced inactivation of cofilin, an actin-binding protein known to sever actin filaments. Thus, the actin cytoskeleton in the PTEN-null cells contains more depolymerized actin, weakening the actin cortex. The combination of apoptotic resistance, the weakening of the actin cortex, and enhanced McTN formation due to PTEN loss may have important consequences for facilitating tumor cell extravasation and efficient adherence in metastatic sites. 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 3122. doi:10.1158/1538-7445.AM2011-3122

Breast cancer is the most frequent malignancy in women, and although many breast cancers are curable via surgery, approximately one quarter maintain a latent and insidious characteristic of slow growth with early metastasis. The loss of the tumor suppressor PTEN has been associated with breast cancer stage, lymph node status, and disease-related death, and the high rate of loss in primary tumors suggests a potential role in initiation and/or progression of the disease. However, specific cellular alterations in human breast epithelium controlled by PTEN inactivation, which lead to an increased metastatic phenotype, remain poorly defined. We have determined that heterozygous and homozygous loss of PTEN in non-tumorigenic mammary epithelial cells (MECs) was insufficient to promote anchorage-independent growth. However, MECs with PTEN loss maintained elevated activation of the PI3K/Akt and MAPK pathways and apoptotic resistance to cell rounding and matrix detatchment. We have also recently determined that PTEN expression loss leads to the production of long, dynamic, tubulin-based membrane protrusions upon detachment, which are increased in frequency, number and length per cell as compared to their isogenic, PTEN-expressing parental counterparts. These novel structures, termed microtentacles (McTNs), are structurally distinct from classical actin-based extensions of adherent cells, persist for days in breast tumor lines that are resistant to anoikis, and aid in the reattachment to matrix or cell monolayers. Therefore, the combination of apoptotic resistance and enhanced McTN formation due to PTEN loss may have important consequences for facilitating tumor cell extravasation and efficient adherence in metastatic sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1123.