MCF-7 cells were treated with 20 M compound c for 4 hours in serum-free media and then suspended. 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. and induce cell death of breast tumor cells [7C11]. Furthermore, metformin is currently being investigated in a number of clinical trials as a potential adjuvant and/or neoadjuvant therapy for breast cancer patients [12]. A number of nonclassical drugs with anti-neoplastic activity have also been shown to activate AMPK as part of their mechanism of action [13]. Therefore, there is currently great interest in developing more selective pharmacological activators of AMPK for clinical use in cancer [14]. Although a great deal of work has been done to study the effects of AMPK on primary tumor formation, its effects on breast malignancy metastasis are still largely unknown. In order to form distant metastases, breast malignancy cells must detach from the extracellular matrix (ECM) and enter into the bloodstream or lymphatic system. Once detached, these CTCs undergo a variety of changes, both molecularly and structurally, to adapt to the new microenvironment. After detachment and survival in the circulation, CTCs must re-attach and arrest at a secondary site [15, 16]. Tumor cell re-attachment is usually a process dependent on stable microtubules [17C21]. Detached breast tumor cells form microtubule-based protrusions, known as microtentacles (McTNs), that aid in CTC aggregation and re-attachment to endothelial cells [19, 22C24]. Therefore, McTNs are crucial structures that may be an important therapeutic target to prevent CTC re-attachment. McTN formation is dependent on the balance LAMA3 antibody of two opposing cytoskeletal forces: the outward pressure of stabilized microtubules and the inward contractile pressure of the actin cortex [19]. Currently two post-translational modifications on alpha tubulin, detyrosination and acetylation, play a significant role in McTN formation [22, 25]. Detyrosination removes the C-terminal tyrosine, exposing a glutamic acid residue, and acetylation takes place around the lysine 40 residue of alpha tubulin by alpha tubulin acetyl-transferase (TAT1/MEC-17) [26, 27]. Both of these modifications are indicators of stabilized microtubules [26C28]. Microtubule stability is associated with greater re-attachment of suspended tumor cells to endothelial monolayers and lung trapping in a murine experimental metastasis model [17, 19, 20, 29]. Increasing glu-tubulin levels, both genetically and pharmacologically, results in greater McTN formation and enhanced suspended cell re-attachment [20, 23, 29, 30]. Elevated acetylated tubulin levels are associated with a higher metastatic phenotype in breast cancer cells and can enhance both McTN formation and re-attachment. In addition, higher levels of acetylated tubulin are enriched in the more aggressive, basal-like subtype of breast cancers and correlate with decreased overall and progression-free survival of breast malignancy patients [25]. Conversely, McTNs are antagonized by the actin cytoskeleton. One major regulator of actin that also plays a significant role in McTN formation is the actin-severing protein, cofilin. Cofilin is usually activated upon dephosphorylation at serine 3, ABT-046 which results in a breakdown of the actin network and increases actin monomers [31]. Activation of cofilin in detached breast epithelial cells promotes McTN formation [24]. There is data to show that AMPK can affect both microtubules and actin in normal epithelial cells [32, 33], but the role of AMPK in regulating the cytoskeleton of breast tumor cells has not yet been investigated. While the metastatic dissemination of CTCs offers a critical windows for cytoskeletal-based therapeutic intervention, microtubule-stabilizing chemotherapies such as taxanes, have cytotoxic side effects and can enhance tumor cell re-attachment [23, 34]. Existing and developing pharmacological AMPK activators that have shown benefit in the primary tumor setting may now also be a potential therapeutic option to decrease the metastatic efficiency of detached breast tumor cells. In this study, we provide a novel role for AMPK in breast malignancy. AMPK inhibition with a pharmacologic inhibitor, compound c, significantly increases microtubule stability and cofilin activation, which leads to increased McTN formation and re-attachment in breast malignancy cell lines. AMPK activation with A-769662 has an opposing effect, significantly reducing McTNs and tumor cell re-attachment by ABT-046 decreasing cofilin activity and microtubule stability. RESULTS A pharmacological approach was utilized to understand the effects of altering the AMPK pathway around the cytoskeletal ABT-046 properties of breast malignancy cells. We selected two different breast malignancy cell lines for this study: a non-metastatic, epithelial cell line, MCF-7 [36] and a more invasive, mesenchymal cell line, BT-549 [37], due.