Microtentacles Research Articles

Functional Analysis of Viable Circulating Tumor Cells from Triple-Negative Breast Cancer Patients Using TetherChip Technology.

Metastasis, rather than the growth of the primary tumor, accounts for approximately 90% of breast cancer patient deaths. Microtentacles (McTNs) formation represents an important mechanism of metastasis. Triple-negative breast cancer (TNBC) is the most aggressive subtype with limited targeted therapies. The present study aimed to isolate viable circulating tumor cells (CTCs) and functionally analyze them in response to drug treatment. CTCs from 20 TNBC patients were isolated and maintained in culture for 5 days. Biomarker expression was identified by immunofluorescence staining and VyCap analysis. Vinorelbine-induced apoptosis was evaluated based on the detection of M30-positive cells. Our findings revealed that the CTC absolute number significantly increased using TetherChips analysis compared to the number of CTCs in patients' cytospins (p = 0.006) providing enough tumor cells for drug evaluation. Vinorelbine treatment (1 h) on live CTCs led to a significant induction of apoptosis (p = 0.010). It also caused a significant reduction in Detyrosinated α-tubulin (GLU), programmed death ligand (PD-L1)-expressing CTCs (p < 0.001), and disruption of McTNs. In conclusion, this pilot study offers a useful protocol using TetherChip technology for functional analysis and evaluation of drug efficacy in live CTCs, providing important information for targeting metastatic dissemination at a patient-individualized level.

Abstract 965: 5-Aza-dC treatment impacts the microtubule network to decrease metastatic potential through reduced cell clustering and attachment

Abstract 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.

Microtentacle Formation in Ovarian Carcinoma.

Simple SummaryOvarian cancer commonly spreads throughout the peritoneal cavity by exfoliation of malignant cells into ascites. Chemoresistance remains an important therapeutic obstacle. Microtentacles (McTNs) are microtubule-based protrusions that may influence the metastatic potential and chemoresistance profile of free-floating cells. In this study, we define the various microtentacle morphologies that can be observed in detached ovarian cancer cells, and their clustering behavior in relationship to histology, α-tubulin post-translational modifications, β-tubulin isotype, modulators of cortical stiffness, and sensitivity to clinically relevant microtubule-targeting agents. McTNs represent a new therapeutic target for this disease, and an understanding of their biology could have implications for the refinement of therapies, including intraperitoneal drug delivery. Background: The development of chemoresistance to paclitaxel and carboplatin represents a major therapeutic challenge in ovarian cancer, a disease frequently characterized by malignant ascites and extrapelvic metastasis. Microtentacles (McTNs) are tubulin-based projections observed in detached breast cancer cells. In this study, we investigated whether ovarian cancers exhibit McTNs and characterized McTN biology. Methods: We used an established lipid-tethering mechanism to suspend and image individual cancer cells. We queried a panel of immortalized serous (OSC) and clear cell (OCCC) cell lines as well as freshly procured ascites and human ovarian surface epithelium (HOSE). We assessed by Western blot β-tubulin isotype, α-tubulin post-translational modifications and actin regulatory proteins in attached/detached states. We studied clustering in suspended conditions. Effects of treatment with microtubule depolymerizing and stabilizing drugs were described. Results: Among cell lines, up to 30% of cells expressed McTNs. Four McTN morphologies (absent, symmetric-short, symmetric-long, tufted) were observed in immortalized cultures as well as ascites. McTN number/length varied with histology according to metastatic potential. Most OCCC overexpressed class III ß-tubulin. OCCC/OSC cell lines exhibited a trend towards more microtubule-stabilizing post-translational modifications of α-tubulin relative to HOSE. Microtubule depolymerizing drugs decreased the number/length of McTNs, confirming that McTNs are composed of tubulin. Cells that failed to form McTNs demonstrated differential expression of α-tubulin- and actin-regulating proteins relative to cells that form McTNs. Cluster formation is more susceptible to microtubule targeting agents in cells that form McTNs, suggesting a role for McTNs in aggregation. Conclusions: McTNs likely participate in key aspects of ovarian cancer metastasis. McTNs represent a new therapeutic target for this disease that could refine therapies, including intraperitoneal drug delivery.

Notch-1 signaling promotes reattachment of suspended cancer cells by cdc42-dependent microtentacles formation.

Circulating tumor cells (CTCs) are associated with a higher risk of metastasis in tumor patients. The adhesion and arrest of CTCs at a secondary site is an essential prerequisite for the occurrence of tumor metastasis. CTC reattachment has shown to be dependent on microtentacle (McTN) formation in vivo. However, the specific molecular mechanism of McTN formation in suspended cancer cells remains largely unclear. Here, we demonstrated that the activation of Notch‐1 signaling triggers McTN formation to facilitate cell reattachment in suspended cell culture conditions. Moreover, molecular mechanistic studies revealed that McTN formation is governed by the balance between microtubule‐driven outgrowth and actomyosin‐driven cell contractility. The activation of Notch‐1 downregulates the acetylation level of microtubules via the Cdc42/HDAC6 pathway, which contributes to microtubule polymerization. Simultaneously, Notch‐1 signaling‐induced Cdc42 activation also reduced phosphorylation of myosin regulatory light chain, leading to cell contractility attenuation. Altogether, these results defined a novel mechanism by which Notch‐1 signaling disturbs the balance between the expansion of microtubules and contraction of the cortical actin, which promotes McTN formation and cell reattachment. Our findings provide a new perspective on the effective therapeutic target to prevent CTC reattachment.

Abstract B68: Characterization of microtentacle phenotype and function in ovarian carcinomas

Abstract Ovarian carcinomas are categorized into five major histotypes, each characterized by distinct differences in grade at diagnosis, presentation with metastatic disease, and clinical responses to treatments. Microtentacles (McTNs), microtubule-based extensions of the plasma membrane, were initially described on cells of nongynecologic primaries with high metastatic potential but have not been extensively explored in ovarian carcinomas. In this study, we investigated whether ovarian cancer cells exhibit McTNs as well as the effect of microtubule-targeted drugs on ovarian cancer McTNs. We analyzed 3 immortalized ovarian surface epithelium cell lines (IOSE), 8 clear-cell (OCCC), 3 low-grade serous (LGS), and 6 high-grade serous (HGS) ovarian cancer cells for McTN phenotype, length, and number, using a novel tethering platform in which cells are suspended but stationary, allowing for analysis via confocal microscopy. Additionally, selected cell lines were also treated with microtubule-targeting agents, including paclitaxel, ixabepilone, vinblastine, and colchicine and the effects on McTN dynamics and functions were analyzed. Tubulin subtype expression and post-translational modifications (PTM) were characterized by Western blot. Unpaired t-tests were used to describe differences between McTN length and number. We observed 4 patterns of McTN expression: absent (A), symmetric-short (SS), symmetric-long (SL), and tufted (T). In some cases, multiple morphologies were observed within the same cell line. LGS and OCCC expressed fewer McTNs per cell than HSC. McTNs were shorter among OCCC compared to HGS; whereas LGS expressed a range of McTN lengths similar to those observed in HGS. We also observed differences in the expression of the PTM between the different ovarian cancer subtypes, including alterations in alpha tubulin-stabilizing modifications such as glu-tubulin and acetylated tubulin, as well as proteins involved in actin cortex stability. Increased acetylated tubulin was associated with increased McTN number and length in HGS cells. Microtubule-destabilizing drugs such as colchicine and vinblastine led to a decrease in McTN formation. Ovarian cancer metastasis typically occurs through shedding of the main tumor into the peritoneal space, and the spread of disease is the leading cause of mortality. Studying McTN function and response to chemotherapeutics allows us to improve our understanding of ovarian cancer metastasis and the effect of microtubule-targeting compounds on the spread of ovarian cancer. Citation Format: Cong (Ava) Fan, Sulan Wu, Jocelyn Reader, Eleanor Claire-Higgins Ory, Cornell Lee, McMillan Ching, Trevor Mathias, Julia Ju, Rachel Lee, Michele Vitolo, Stuart Martin, Christopher Jewell, Amy Fulton, Gautam Rao, Mark Carey, Dana M. Roque. Characterization of microtentacle phenotype and function in ovarian carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B68.

A mode of cell adhesion and migration facilitated by CD44-dependent microtentacles

The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.

Extracting microtentacle dynamics of tumor cells in a non-adherent environment.

During metastasis, tumor cells dynamically change their cytoskeleton to traverse through a variety of non-adherent microenvironments, including the vasculature or lymphatics. Due to the challenges of imaging drift in non-adhered tumor cells, the dynamic cytoskeletal phenotypes are poorly understood. We present a new approach to analyze the dynamic cytoskeletal phenotypes of non-adhered cells that support microtentacles (McTNs), which are cell surface projections implicated in metastatic reattachment. Combining a recently-developed cell tethering method with a novel image analysis framework allowed McTN attribute extraction. Full cell outlines, number of McTNs, and distance of McTN tips from the cell body boundary were calculated by integrating a rotating anisotropic filtering method for identifying thin features with retinal segmentation and active contour algorithms. Tethered cells behave like free-floating cells; however tethering reduces cell drift and improves the accuracy of McTN measurements. Tethering cells does not significantly alter McTN number, but rather allows better visualization of existing McTNs. In drug treatment experiments, stabilizing tubulin with paclitaxel significantly increases McTN length, while destabilizing tubulin with colchicine significantly decreases McTN length. Finally, we quantify McTN dynamics by computing the time delay autocorrelations of 2 composite phenotype metrics (cumulative McTN tip distance, cell perimeter:cell body ratio). Our automated analysis demonstrates that treatment with paclitaxel increases total McTN amount and colchicine reduces total McTN amount, while paclitaxel also reduces McTN dynamics. This analysis method enables rapid quantitative measurement of tumor cell drug responses within non-adherent microenvironments, using the small numbers of tumor cells that would be available from patient samples.

Abstract 3978: Lipid tethering to enable real-time imaging of breast tumor cell cytoskeletal dynamics and rapid drug testing in free-floating metastatic microenvironments

Abstract Free-floating tumor cells located in the peripheral circulation of cancer patients, known as circulating tumor cells (CTCs), play an important role in cancer and have become key targets for studying metastasis. The presence of CTCs in the bloodstream or lymphatics correlates with decreased cancer patient survival and provides a minimally invasive method to study disease progression and treatment response. Understanding the molecular characteristics and functional properties of CTCs have been impeded by the challenges of imaging CTCs under conditions that model the free-floating microenvironments of the circulation. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells within a microfluidic device while maintaining their free-floating character. We use cytophobic polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membrane. This approach enables high-resolution time-lapse microscopy of McTNs on viable free-floating tumor cells and real-time analysis of dynamic cellular features. In addition, tethering makes it now possible to rapidly measure drug responses of tumor cells in free-floating microenvironments to select effective therapies and avoid drugs that could inadvertently increase metastasis. The ability to image tumor cells in the absence of ECM attachment can immensely enhance our understanding of CTCs under conditions that better recapitulate the free-floating microenvironments that tumor cells encounter during metastasis. Citation Format: Kristi Riti Chakrabarti, James Andorko, Rebecca Whipple, Peipei Zhang, Elisabeth Sooklal, Christopher Jewell, Stuart Martin. Lipid tethering to enable real-time imaging of breast tumor cell cytoskeletal dynamics and rapid drug testing in free-floating metastatic microenvironments. [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 3978.

Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response

Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis.

Pharmacologic regulation of AMPK in breast cancer affects cytoskeletal properties involved with microtentacle formation and re-attachment.

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.

α-Tubulin acetylation elevated in metastatic and basal-like breast cancer cells promotes microtentacle formation, adhesion, and invasive migration.

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.

Abstract 3160: ROCK inhibition promotes microtentacle formation and reattachment of breast cancer cells

Abstract 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

Curcumin Targets Breast Cancer Stem–like Cells with Microtentacles That Persist in Mammospheres and Promote Reattachment

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.

Interplay of Stem Cell Characteristics, EMT, and Microtentacles in Circulating Breast Tumor Cells.

Metastasis, not the primary tumor, is responsible for the majority of breast cancer-related deaths. Emerging evidence indicates that breast cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis. CTCs with both CSC and EMT characteristics have recently been identified in the bloodstream of patients with metastatic disease. Breast CSCs have elevated tumorigenicity required for metastatic outgrowth, while EMT may promote CSC character and endows breast cancer cells with enhanced invasive and migratory potential. Both CSCs and EMT are associated with a more flexible cytoskeleton and with anoikis-resistance, which help breast carcinoma cells survive in circulation. Suspended breast carcinoma cells produce tubulin-based extensions of the plasma membrane, termed microtentacles (McTNs), which aid in reattachment. CSC and EMT-associated upregulation of intermediate filament vimentin and increased detyrosination of α-tubulin promote the formation of McTNs. The combined advantages of CSCs and EMT and their associated cytoskeletal alterations increase metastatic efficiency, but understanding the biology of these CTCs also presents new therapeutic targets to reduce metastasis.

Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition

IntroductionDetyrosinated 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.MethodsUsing 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.ResultsSesquiterpene 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.ConclusionsOur 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.

Abstract 3772: The role of microtentacles in the metastatic potential of breast tumor stem cells.

Abstract 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

Abstract 1480: The effect of Metformin on microtentacle formation and stabilization in breast cancer cells.

Abstract 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

Abstract 276: Microtentacle stabilization during TGFβ-induced EMT as a new therapeutic target to reduce circulating tumor cell metastasis.

Abstract 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

Abstract 2627: Acetylation of α-tubulin contributes to microtentacle formation and re-attachment in suspended breast tumor cells.

Abstract 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

Loss of PTEN induces microtentacles through PI3K-independent activation of cofilin

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.