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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.

Supplementary Figure Legends.

Supplementary Figure S2. K40R α-tubulin does not affect proliferation in MDA-MB-231 or BT-549 cells but decreases McTN frequency under transient transfection conditions.

Supplementary Table S1. Patient characteristics for survival analysis.

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer. Cancer Res; 75(1); 203–15. ©2014 AACR.

Supplementary Figure S4. Patient ages and pathological diagnosis for 144 matched patient samples.

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

Supplementary Figure S3. αTAT1-GFP or GFP control transfection does not affect the α-tubulin network or proliferation.

Supplementary Figure S1. Detyrosinated α-tubulin is not elevated in metastatic breast tumor cell lines, while acetylated α-tubulin is maintained under suspended conditions and correlates with more numerous McTNs in metastatic breast cancer cells.

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.

Supplementary Figure S5. High acetylation is linked to the basal-like subtype and reduced progression-free and overall survival in patients.

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.

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.

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer. Cancer Res; 75(1); 203–15. ©2014 AACR.

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.

Both the G2 chromosomal radiosensitivity assay and allelic differences in TP53 codon-72 have been associated with cancer predisposition. The relationship between the two endpoints was determined in 56 human EBV-transformed lymphoblastoid cell lines. Although there were overlapping distributions of sensitivity for the different genotypes, cell lines that were homozygous for the proline coding allele were more likely to be resistant to chromatid break formation than those containing two arginine coding alleles, whereas cell lines expressing both the proline and arginine codon were either resistant like proline–proline lines or sensitive like arginine–arginine lines. The results support an important role of the TP53 codon-72 polymorphism in modifying G2-chromosome radiosensitivity. Distinguishing the effect of TP53 codon-72 variations from other modifiers of G2-chromosome radiosensitivity might aid in identifying new markers of cancer risk. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc.

To characterize the radioadaptive response in the human lymphoblastoid cell model TK6, and determine: (i) Whether repeated low dose exposures are more effective than single acute exposures in inducing resistance, (ii) the time-course for induction and loss of resistance following chronic exposures, and (iii) the effect of TP53 deletion or BCL2 over-expression on the induction of an adaptive response.TK6, a human B-lymphoblastoid cell line, TK6-BCL2, a TK6 line that over-expresses BCL2 and is resistant to radiation-induced apoptosis, and NH32, a TP53 knockout of TK6 that is also resistant to apoptosis were studied. Cells were exposed to chronic, daily doses of 10 cGy given over 1 -21 days before being challenged with 1 -5 Gy exposures. Cell survival and chromatid break induction following high dose challenge were used to evaluate adaptive radiation responses.Exposure to 10 cGy gamma rays induced resistance to killing and chromosome break induction in TK6 cells, but not in either TK6-BCL2 or NH32 cells. Resistance in TK6 was observed 4 h after exposure, and cells remained resistant for about 48 h. Maximal resistance was induced by a single 10 cGy dose. Repeated 10 cGy exposures had no additional effect on radiation sensitivity, except to maintain the induced radioresistance.An adaptive response is maximally and rapidly induced by a single low dose exposure in TK6 cells, and it has a limited lifespan. Induction of an adaptive response in TK6 cells can be abrogated by either TP53 loss or BCL2 over-expression. The characteristics of induced resistance in TK6 cells suggest that alterations in TP53-dependent apoptotic responses may be one mechanism for resistance.

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

Circulating tumor cells (CTCs) seed distant and recurrent metastases, the leading cause of breast cancer-related death. However, few laboratories are investigating cytoskeletal properties of CTCs or are targeting CTCs directly. We have shown that detached epithelial cells produce microtubule-based protrusions called microtentacles (McTNs) that facilitate CTC re-attachment. These protrusions can be stabilized by microtubule-associated proteins or post-translational modifications of α-tubulin. Acetylation is a post-translational modification that occurs on Lysine 40 (K40) of α-tubulin by a recently identified α-tubulin acetyltransferase: ATAT1. This modification is understudied in breast cancer and has not been previously examined in detached tumor cells. It is also clinically relevant, since HDAC inhibitors and microtubule-stabilizing drugs increase acetylation of α-tubulin in vivo. We have found that α-tubulin acetylation increases with metastatic potential in a panel of breast tumor cell lines and localizes to McTN protrusions in suspended metastatic cells. In tumor cells with high endogenous tubulin acetylation, immunofluorescence showed that cells transiently transfected with a non-acetylatable K40R α-tubulin mutant had fewer acetylated microtubules compared to adjacent, non-transfected cells. Functional tests in cells stably expressing K40R demonstrated that decreased α-tubulin acetylation reduced microtentacle formation. Electrical impedance also revealed decreased tumor cell reattachment in cells stably expressing the K40R mutant, compared to wild-type controls. In tumor cells with low endogenous α-tubulin acetylation, transient transfection of the acetyltransferase ATAT1 significantly increased acetylation of α-tubulin, compared to controls on immunoblot and compared to adjacent/non-transfected cells with immunofluorescence. Live-cell fluorescence imaging showed transient ATAT1 overexpression also significantly increased microtentacle frequency. Suspended immunofluorescence showed acetylated α-tubulin localized to McTNs in ATAT1 overexpressing cells but not in controls. ATAT1 overexpression also increased tumor cell reattachment. Since clinical tumor imaging requires a foci of more than 5 million tumor cells, current interpretations of drug effects are limited to large changes in tumor size rather than effects on metastatic dissemination. The ability of elevated α-tubulin acetylation to increase microtentacles and promote tumor cell reattachment emphasizes the importance of understanding how α-tubulin acetylation influences CTC metastasis so therapies aimed at tumor growth do not inadvertently elevate metastatic risk. Further investigation into matched metastatic breast tumor patient samples will reveal the clinical relevance of this previously understudied modification. Citation Format: Amanda E. Boggs, Michele I. Vitolo, Rebecca A. Bettes, Jana Slovic, Monica S. Charpentier, Stuart S. Martin. Acetylation of α-tubulin contributes to microtentacle formation and re-attachment in suspended breast tumor 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 2627. doi:10.1158/1538-7445.AM2013-2627

Metastasis is one of the main causes of mortality in women with breast cancer. Epithelial-to-mesenchymal transition (EMT) has emerged to be a key mediator of breast cancer metastasis. It has previously been shown that EMT can stabilize and increase microtentacles (McTNs) in breast cancer cell lines and enhance their metastatic potential. McTNs are dynamic, microtubule-enriched plasma membrane extensions that are stabilized with the exposure of a carboxy-terminal glutamic acid residue in alpha-tubulin (Glu-tubulin). McTN stabilization has been shown to promote tumor cell aggregation and reattachment in vitro. In this study, we investigated the effect of EMT suppression on McTN formation and stabilization in breast cancer cells by the dimethyl biguanide, Metformin. While it is widely used to treat type 2 diabetes, Metformin has more recently been shown to inhibit EMT in multiple breast cancer cell lines. We show here that Metformin decreased mesenchymal markers, such as N-cadherin and vimentin, in a dose-dependent manner in MDA-MB-231 breast cancer cells. Confocal and fluorescent microscopy was used to visualize and quantify McTN formation in these cells. There was a significant decrease in McTNs in MDA-MB-231 cells treated with 5mM Metformin for 24 hours. We correlated the decrease in the number of McTNs with a decrease in Glu-tubulin protein levels, which suggests that Metformin also inhibits McTN stabilization. The decrease in McTN formation and stabilization was also associated with diminished tumor cell reattachment in vitro. In addition, Metformin was able to protect MDA-MB-231 cells from increased McTN formation induced by the microtubule stabilizing agent, Taxol. Metformin's mechanism of action on breast cancer cells to elicit these cytoskeletal modifications has yet to be elucidated. However, these results suggest a role for Metformin at multiple stages of tumor progression to protect breast cancer patients from clinical spread of disease. Citation Format: Kristi Chakrabarti, Rebecca A. Whipple, Amanda E. Boggs, Monica S. Charpentier, Jana Slovic, Michele I. Vitolo, Stuart S. Martin. The effect of Metformin on microtentacle formation and stabilization in breast cancer 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 1480. doi:10.1158/1538-7445.AM2013-1480

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