Estrogen, progesterone, and HER2 receptor-negative triple-negative breast cancers encompass the most clinically challenging subtype for which targeted therapeutics are lacking. We find that triple-negative tumors exhibit elevated MYC expression, as well as altered expression of MYC regulatory genes, resulting in increased activity of the MYC pathway. In primary breast tumors, MYC signaling did not predict response to neoadjuvant chemotherapy but was associated with poor prognosis. We exploit the increased MYC expression found in triple-negative breast cancers by using a synthetic-lethal approach dependent on cyclin-dependent kinase (CDK) inhibition. CDK inhibition effectively induced tumor regression in triple-negative tumor xenografts. The proapoptotic BCL-2 family member BIM is up-regulated after CDK inhibition and contributes to this synthetic-lethal mechanism. These results indicate that aggressive breast tumors with elevated MYC are uniquely sensitive to CDK inhibitors.

In cancer progression, carcinoma cells gain invasive behavior through a loss of epithelial characteristics and acquisition of mesenchymal properties, a process that can lead to epithelial–mesenchymal transition (EMT). TGF-β is a potent inducer of EMT, and increased TGF-β signaling in cancer cells is thought to drive cancer-associated EMT. Here, we examine the physiological requirement for mTOR complex 2 (mTORC2) in cells undergoing EMT. TGF-β rapidly induces mTORC2 kinase activity in cells undergoing EMT, and controls epithelial cell progression through EMT. By regulating EMT-associated cytoskeletal changes and gene expression, mTORC2 is required for cell migration and invasion. Furthermore, inactivation of mTORC2 prevents cancer cell dissemination in vivo. Our results suggest that the mTORC2 pathway is an essential downstream branch of TGF-β signaling, and represents a responsive target to inhibit EMT and prevent cancer cell invasion and metastasis.

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts. Calcium plays a primary role in regulating heart function. During each heartbeat, calcium ions cross the membrane of individual cardiac muscle cells and trigger a rapid increase of calcium within the cell (called the calcium transient). Calcium causes the muscle cells to contract and determines the strength of the overall heartbeat. Each cardiac muscle cell has many small tubular-like membrane invaginations known as T-tubules where calcium channels localize, allowing calcium ions to enter and immediately encounter intracellular calcium release organelles. While this organization is well described, it is not known how calcium channels localize to T-tubule membrane. Here we show that in human and mouse heart cells, a membrane scaffolding protein known as BIN1 is localized together with calcium channels at T-tubules. Using high-resolution live cell microscopy, we found that microtubules, which are necessary for calcium channel delivery to the membrane, are also tethered by BIN1. Loss of BIN1 in cardiac cells impairs delivery of calcium channels to the membrane and diminishes the intracellular calcium transient. According to this model, microtubules function as highways that carry newly synthesized calcium channels to BIN1-containing membrane. Once tethered to T-tubules by BIN1, the microtubules can deliver their calcium channel cargo. We postulate that this calcium channel delivery pathway is important to the regulation of cardiac calcium signaling and beat-to-beat cardiac function.

Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.

Background— Heart disease is a leading cause of mortality throughout the world. Tissue damage from vascular occlusive events results in the replacement of contractile myocardium by nonfunctional scar tissue. The potential of new technologies to regenerate damaged myocardium is significant, although cell-based therapies must overcome several technical barriers. One possible cell-independent alternative is the direct administration of small proteins to damaged myocardium. Methods and Results— Here we show that the secreted signaling protein stromal cell–derived factor-1α (SDF-1α), which activates the cell-survival factor protein kinase B (PKB/Akt) via the G protein–coupled receptor CXCR4, protected tissue after an acute ischemic event in mice and activated Akt within endothelial cells and myocytes of the heart. Significantly better cardiac function than in control mice was evident as early as 24 hours after infarction as well as at 3, 14, and 28 days after infarction. Prolonged survival of hypoxic myocardium was followed by an increase in levels of vascular endothelial growth factor protein and neoangiogenesis. Consistent with improved cardiac function, mice exposed to SDF-1α demonstrated significantly decreased scar formation than control mice. Conclusion— These findings suggest that SDF-1α may serve a tissue-protective and regenerative role for solid organs suffering a hypoxic insult.

During each heartbeat, intercellular electrical coupling via connexin43 (Cx43) gap junctions enables synchronous cardiac contraction. In failing hearts, impaired Cx43 trafficking reduces gap junction coupling, resulting in arrhythmias. Here we report that internal translation within Cx43 (GJA1) mRNA occurs, resulting in truncated isoforms that autoregulate Cx43 trafficking. We find that at least four truncated Cx43 isoforms occur in the human heart, with a 20 kDa isoform predominating. In-frame AUG codons within GJA1 mRNA are the translation initiation sites and their ablation arrests trafficking of full-length Cx43. The 20 kDa isoform is sufficient to rescue this trafficking defect in trans, suggesting it as a trafficking chaperone for Cx43. Limiting cap-dependent translation through inhibition of mTOR enhances truncated isoform expression, increasing Cx43 gap junction size. The results suggest that internal translation is a mechanism of membrane protein autoregulation and a potent target for therapies aimed at restoring normal electrical coupling in diseased hearts.