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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.

PDF file - 103K, Vehicle-treated BT-549s form significantly more mammospheres than BT-549s treated with 50microM curcumin.

AVI file - 5792K, Live cell confocal imaging of HMLE mammosphere stained with CellMask Orange (red) and transduced with GFP-membrane (green) suspended over a glass surface. Three-dimensional computer-rendered image reveals McTNs from the GFP-membrane cell encircling other cells in the mammosphere.

AVI file - 4552K, Live cell confocal imaging of HMLE mammosphere stained with CellMask Orange (red) and transduced with GFP-membrane (green) suspended over a glass surface. Cross-sections reveal McTNs from the GFP-membrane cell encircling other cells in the mammosphere.

AVI file - 1306K, Live cell confocal imaging of a HMLE mammosphere stained with CellMask Orange revealing dynamic microtentacles.

MOV file - 1782K, Live cell confocal imaging of BT-549 mammosphere stained with CellMask Orange (red) and transduced with GFP-membrane (green) suspended over a glass surface. Three-dimensional computer-rendered image reveals McTNs from the GFP-membrane cell encircling other cells in the mammosphere.

MOV file - 1194K, Live cell confocal imaging of BT-549 mammosphere stained with CellMask Orange (red) and transduced with GFP-membrane (green) suspended over a glass surface. Cross-sections reveal McTNs from the GFP-membrane cell encircling other cells in the mammosphere.

PDF file - 110K, Cellular viability assay reveals no significant differences in viability between HMLE stem-like and non-stem-like subpopulations upon treatment with colchicine.

Dendritic cells (DCs) are increasingly important for research and clinical use but obtaining sufficient numbers of dendritic cells is a growing challenge. We systemically investigated the effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) in MicroDEN, a perfusion-based culture system, as well as 6-well plates. Cell surface markers and the ability of the iDCs to induce proliferation of allogeneic T cells were examined. The data shows a strong relationship between iDC phenotype, specifically CD80/83/86 expression, and T cell proliferation. MicroDEN generated iDCs proved better than well plate generated iDCs at inducing T cell proliferation within the 200k-600k MO/cm(2) seeding density range studied. We attribute this to perfusion in MicroDEN which supplies fresh differentiation medium continuously to the differentiating MOs while concurrently removing depleted medium and toxic byproducts of cellular respiration. MicroDEN generated fewer iDCs on a normalized basis than the well plates at lower MO seeding densities but generated equivalent numbers of iDCs at 600k MO seeding density. These results demonstrate that MicroDEN is capable of generating greater numbers of iDCs with less manual work than standard well plate culture and the MicroDEN generated iDCs have greater ability to induce T cell proliferation.

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.

RUNX2 and SOX9 are two pivotal transcriptional regulators of chondrogenesis. It has been demonstrated that RUNX2 and SOX9 physically interact; RUNX2 transactivation may be inhibited by SOX9. In addition, RUNX2 exerts reciprocal inhibition on SOX9 transactivity. Epigenetic control of gene expression plays a major role in the alternative differentiation fates of stem cells; in particular, it has been reported that SOX9 can promote the expression of miRNA (miR)-204. Our aim was therefore to investigate the miR-204-5p role during chondrogenesis and to identify the relationship between this miR and the transcription factors plus downstream genes involved in chondrogenic commitment and differentiation. To evaluate the role of miR-204 in chondrogenesis, we performed in vitro transfection experiments by using Mesenchymal Stem Cells (MSCs). We also evaluated miR-204-5p expression in zebrafish models (adults and larvae). By silencing miR-204 during the early differentiation phase, we observed the upregulation of SOX9 and chondrogenic related genes compared to controls. In addition, we observed the upregulation of COL1A1 (a RUNX2 downstream gene), whereas RUNX2 expression of RUNX2 was slightly affected compared to controls. However, RUNX2 protein levels increased in miR-204-silenced cells. The positive effects of miR204 silencing on osteogenic differentiation were also observed in the intermediate phase of osteogenic differentiation. On the contrary, chondrocytes’ maturation was considerably affected by miR-204 downregulation. In conclusion, our results suggest that miR-204 negatively regulates the osteochondrogenic commitment of MSCs, while it positively regulates chondrocytes’ maturation.

Dendritic cells (DCs) are an indispensable part of studying human responses that are important for protective immunity against cancer and infectious diseases as well as prevention of autoimmunity and transplant rejection. These cells are also key elements of personalized vaccines for cancer and infectious diseases. Despite the vital role of DCs in both clinical and basic research contexts, methods for obtaining these cells from individuals remains a comparatively under-developed and inefficient process. DCs are present in very low concentrations (

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.

Activation of genes promoting aerobic glycolysis and suppression of mitochondrial oxidative phosphorylation is one of the hallmarks of cancer. The RUNX2 transcription factor mediates breast cancer (BC) metastasis to bone and is regulated by glucose availability. But, the mechanisms by which it regulates glucose metabolism and promotes an oncogenic phenotype are not known. RUNX2 expression in luminal BC cells correlated with lower estrogen receptor-α (ERα) levels, anchorage-independent growth, expression of glycolytic genes, increased glucose uptake, and sensitivity to glucose starvation, but not to inhibitors of oxidative phosphorylation. Conversely, RUNX2 knockdown in triple-negative BC cells inhibited mammosphere formation and glucose dependence. RUNX2 knockdown resulted in lower LDHA, HK2, and GLUT1 glycolytic gene expression, but upregulation of pyruvate dehydrogenase-A1 (PDHA1) mRNA and enzymatic activity, which was consistent with lower glycolytic potential. The NAD-dependent histone deacetylase, SIRT6, a known tumor suppressor, was a critical regulator of these RUNX2-mediated metabolic changes. RUNX2 expression resulted in elevated pAkt, HK2, and PDHK1 glycolytic protein levels that were reduced by ectopic expression of SIRT6. RUNX2 also repressed mitochondrial oxygen consumption rates (OCR), a measure of oxidative phosphorylation (respiration). Overexpression of SIRT6 increased respiration in RUNX2-positive cells, but knockdown of SIRT6 in cells expressing low RUNX2 decreased respiration. RUNX2 repressed SIRT6 expression at both the transcriptional and post-translational levels and endogenous SIRT6 expression was lower in malignant BC tissues or cell lines that expressed high levels of RUNX2. These results support a hypothesis whereby RUNX2-mediated repression of the SIRT6 tumor suppressor regulates metabolic pathways that promote BC progression.

// 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.

The dynamic balance between microtubule extension and actin contraction regulates mammalian cell shape, division, and motility, which has made the cytoskeleton an attractive and very successful target for cancer drugs. Numerous compounds in clinical use to reduce tumor growth cause microtubule breakdown (vinca alkaloids, colchicine-site, and halichondrins) or hyperstabilization of microtubules (taxanes and epothilones). However, both of these strategies indiscriminately alter the assembly and dynamics of all microtubules, which causes significant dose-limiting toxicities on normal tissues. Emerging data are revealing that posttranslational modifications of tubulin (detyrosination, acetylation) or microtubule-associated proteins (Tau, Aurora kinase) may allow for more specific targeting of microtubule subsets, thereby avoiding the broad disruption of all microtubule polymerization. Developing approaches to reduce tumor cell migration and invasion focus on disrupting actin regulation by the kinases SRC and ROCK. Because the dynamic balance between microtubule extension and actin contraction also regulates cell fate decisions and stem cell characteristics, disrupting this cytoskeletal balance could yield unexpected effects beyond tumor growth. This review will examine recent data demonstrating that cytoskeletal cancer drugs affect wound-healing responses, microtentacle-dependent reattachment efficiency, and stem cell characteristics in ways that could affect the metastatic potential of tumor cells, both beneficially and detrimentally. Clin Cancer Res; 21(23); 5209–14. ©2015 AACR.

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

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.

Breast cancer remains the most common cancer and second-leading cause of cancer-related death in women, with metastatic disease carrying a particularly poor prognosis. Despite advances with chemotherapeutic drugs aimed at tumor growth, we do not fully understand the effects of these drugs on the metastatic process. Histone deacetylase inhibitors (HDACi) are novel epigenetic therapies that induce cancer cell death through various mechanisms, including apoptosis, cell-cycle arrest and antiangiogenesis. Clinical trials investigating HDACi use in solid tumors are ongoing but have yielded mixed results. To date, very few studies have examined how HDACi influence the metastatic potential of breast tumor cells. Since HDAC enzymes act on both histone and non-histone targets, HDACi drugs cause dramatic increases in non-histone protein acetylation, including acetylation of alpha-tubulin. A well-known marker of stable microtubules, tubulin acetylation increases in metastatic breast tumors and predicts poor patient survival. However, the effects of HDACi on tubulin-related cytoskeletal dynamics of tumor cells have not been previously examined. The current study investigates whether the use of HDACi in breast cancer cells could promote metastatic phenotypes. Clinically-relevant HDAC pan-inhibitors that target multiple Class I HDAC enzymes (Entinostat) or both Class I and Class II (FDA-approved Vorinostat) yielded strong and rapid increases in the migration of both MCF-7 and Bt-549 breast cancer cells. In addition, Vorinostat and Entinostat were each able to strongly induce histone acetylation, while only Vorinostat induced tubulin acetylation. To determine if the increased tubulin acetylation induced by HDACi drugs was sufficient to induce tumor cell migration, we used an isotype-specific HDACi (Tubastatin) that selectively targets the Class II HDAC6 and therefore can increase tubulin acetylation. At doses which strongly induce tubulin acetylation, Tubastatin did not increase tumor cell migration or other tubulin-dependent cancer cell processes, including reattachment and microtentacle formation. Vorinostat and Entinostat therefore induce tumor cell migration through a tubulin-independent mechanism, indicating that more precise targeting of HDAC enzymes could help avoid therapeutic approaches that induce metastatic phenotypes. Citation Format: Lindsay K. Hessler, Amanda E. Boggs, Rebecca A. Whipple, Michele I. Vitolo, Kristi R. Chakrabarti, Lekhana Bhandary, Keyata N. Thompson, Stuart S. Martin. HDAC inhibition promotes tubulin-independent cancer cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-342.

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

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