Amanda E. Boggs

Physical confinement alters tumor cell adhesion and migration phenotypes

Cell migration on planar surfaces is driven by cycles of actin protrusion, integrin‐mediated adhesion, and myosin‐mediated contraction; however, this mechanism may not accurately describe movement in 3‐dimensional (3D) space. By subjecting cells to restrictive 3D environments, we demonstrate that physical confinement constitutes a biophysical stimulus that alters cell morphology and suppresses mesenchymal motility in human breast carcinoma (MDA‐MB‐231). Dorsoventral polarity, stress fibers, and focal adhesions are markedly attenuated by confinement. Inhibitors of myosin, Rho/ROCK, or β1‐integrins do not impair migration through 3‐μm‐wide channels (confinement), even though these treatments repress motility in 50‐μm‐wide channels (unconfined migration) by ≥50%. Strikingly, confined migration persists even when F‐actin is disrupted, but depends largely on microtubule (MT) dynamics. Interfering with MT polymerization/depolymerization causes confined cells to undergo frequent directional changes, thereby reducing the average net displacement by ≥80% relative to vehicle controls. Live‐cell EB1‐GFP imaging reveals that confinement redirects MT polymerization toward the leading edge, where MTs continuously impact during advancement of the cell front. These results demonstrate that physical confinement can induce cytoskeletal alterations that reduce the dependence of migrating cells on adhesion‐contraction force coupling. This mechanism may explain why integrins can exhibit reduced or altered function during migration in 3D environments.—Balzer, E. M., Tong, Z., Paul, C. D., Hung, W.‐C., Stroka, K. M., Boggs, A. E., Martin, S. S., Konstantopoulos, K. Physical confinement alters tumor cell adhesion and migration phenotypes. FASEB J. 26, 4045–4056 (2012). www.fasebj.org

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.

c-Src differentially regulates the functions of microtentacles and invadopodia

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 the expression of a constitutively active c-Src kinase, and repressed by the 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 ECM. In addition, 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.

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.

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.

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.

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.

The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence

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.

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

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.

Abstract A32: Cell dormancy and tumorigenicity due to PTEN loss

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