Kristi R. Chakrabarti

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.

Molecular Pathways: New Signaling Considerations When Targeting Cytoskeletal Balance to Reduce Tumor Growth

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

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.

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.

Single-Cell Tracking of Breast Cancer Cells Enables Prediction of Sphere Formation from Early Cell Divisions

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

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 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.

Abstract 2161: Activation of the MAPK pathway in combination with PTEN loss leads to aggressive primary tumor formation

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

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

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. Metformin9s 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