Hydrogen Peroxide Induces α-Tubulin Detyrosination and Acetylation and Impacts Breast Cancer Metastatic Phenotypes.

Levels of hydrogen peroxide are highly elevated in the breast tumor microenvironment compared to normal tissue. Production of hydrogen peroxide is implicated in the mechanism of action of many anticancer therapies. Several lines of evidence suggest hydrogen peroxide mediates breast carcinogenesis and metastasis, though the molecular mechanism remains poorly understood. This study elucidates the effects of exposure to elevated hydrogen peroxide on non-tumorigenic MCF10A mammary epithelial cells, tumorigenic MCF7 cells, and metastatic MDA-MB-231 breast cancer cells. Hydrogen peroxide treatment resulted in a dose- and time-dependent induction of two α-tubulin post-translational modifications-de-tyrosination and acetylation-both of which are markers of poor patient prognosis in breast cancer. Hydrogen peroxide induced the formation of tubulin-based microtentacles in MCF10A and MDA-MB-231 cells, which were enriched in detyrosinated and acetylated α-tubulin. However, the hydrogen peroxide-induced microtentacles did not functionally promote metastatic phenotypes of cellular reattachment and homotypic cell clustering. These data establish for the first time that microtentacle formation can be separated from the functions to promote reattachment and clustering, which indicates that there are functional steps that remain to be identified. Moreover, signals in the primary tumor microenvironment may modulate α-tubulin post-translational modifications and induce microtentacles; however, the functional consequences appear to be context-dependent.

Metastatic breast cancer cells have reduced calcium and actin response after ATP-P2Y2 signaling.

The tumor microenvironment and wound healing after injury, both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it’s altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cellular junctions, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation could be blunted using a P2Y2 antagonist, which also prevented actin mobilization in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. STATEMENT OF SIGNIFICANCE: This work shows non-tumorigenic breast epithelial cells increase intracellular Ca 2+ after ATP-P2Y2 signaling and re-localize actin, while metastatic cells lack this response, due to decreased P2Y2 expression, which correlates with poorer survival.

OBSCN restoration via OBSCN-AS1 long-noncoding RNA CRISPR-targeting suppresses metastasis in triple-negative breast cancer.

Mounting evidence implicates the giant, cytoskeletal protein obscurin (720 to 870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work has shown that the sole loss of OBSCN from normal breast epithelial cells increases survival and chemoresistance, induces cytoskeletal alterations, enhances cell migration and invasion, and promotes metastasis in the presence of oncogenic KRAS. Consistent with these observations, analysis of Kaplan-Meier Plotter datasets reveals that low OBSCN levels correlate with significantly reduced overall and relapse-free survival in breast cancer patients. Despite the compelling evidence implicating OBSCN loss in breast tumorigenesis and progression, its regulation remains elusive, limiting any efforts to restore its expression, a major challenge given its molecular complexity and gigantic size (~170 kb). Herein, we show that OBSCN-Antisense RNA 1 (OBSCN-AS1), a novel nuclear long-noncoding RNA (lncRNA) gene originating from the minus strand of OBSCN, and OBSCN display positively correlated expression and are downregulated in breast cancer biopsies. OBSCN-AS1 regulates OBSCN expression through chromatin remodeling involving H3 lysine 4 trimethylation enrichment, associated with open chromatin conformation, and RNA polymerase II recruitment. CRISPR-activation of OBSCN-AS1 in triple-negative breast cancer cells effectively and specifically restores OBSCN expression and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Collectively, these results reveal the previously unknown regulation of OBSCN by an antisense lncRNA and the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, which may be used as prognostic biomarkers and/or therapeutic targets for metastatic breast cancer.

Elevation of Cytoplasmic Calcium Suppresses Microtentacle Formation and Function in Breast Tumor Cells.

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.

Polarized NHE1 and SWELL1 regulate migration direction, efficiency and metastasis.

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. The physiological relevance of the Osmotic Engine Model and the identity of molecules mediating cell rear shrinkage remain elusive. Here, we demonstrate that NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, cell dissemination from spheroids and confined migration. SWELL1 polarization confers migration direction and efficiency, as predicted mathematically and determined experimentally via optogenetic spatiotemporal regulation. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal in SWELL1-expressing, but not SWELL1-knockdown, cells. Efficient cell reversal also requires Cdc42, which controls NHE1 repolarization. Dual NHE1/SWELL1 knockdown inhibits breast cancer cell extravasation and metastasis in vivo, thereby illustrating the physiological significance of the Osmotic Engine Model.

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling.

Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

Microtubule disruption reduces metastasis more effectively than primary tumor growth.

Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.

Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation.

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 µm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas.

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Partial thermal imidization of polyelectrolyte multilayer cell tethering surfaces (TetherChip) enables efficient cell capture and microtentacle fixation for circulating tumor cell analysis.

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.

A microfluidic assay for the quantification of the metastatic propensity of breast cancer specimens.

The challenge of predicting which patients with breast cancer will develop metastases leads to the overtreatment of patients with benign disease and to the inadequate treatment of aggressive cancers. Here, we report the development and testing of a microfluidic assay that quantifies the abundance and proliferative index of migratory cells in breast cancer specimens, for the assessment of their metastatic propensity and for the rapid screening of potential antimetastatic therapeutics. On the basis of the key roles of cell motility and proliferation in cancer metastasis, the device accurately predicts the metastatic potential of breast cancer cell lines and of patient-derived xenografts. Compared with unsorted cancer cells, highly motile cells isolated by the device exhibited similar tumourigenic potential but markedly increased metastatic propensity in vivo. RNA sequencing of the highly motile cells revealed an enrichment of motility-related and survival-related genes. The approach might be developed into a companion assay for the prediction of metastasis in patients and for the selection of effective therapeutic regimens.

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

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.

Real-time scratch assay reveals mechanisms of early calcium signaling in breast cancer cells in response to wounding.

Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500µm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

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.

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.

ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.

Cyclin D1 represses gluconeogenesis via inhibition of the transcriptional coactivator PGC1α.

Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1-mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

Vulnerability of the developing heart to oxygen deprivation as a cause of congenital heart defects.

BACKGROUND: The heart develops under reduced and varying oxygen concentrations, yet there is little understanding of oxygen metabolism in the normal and mal-development of the heart. Here we used a novel reagent, the ODD-Luc hypoxia reporter mouse (oxygen degradation domain, ODD) of Hif-1α fused to Luciferase (Luc), to assay the activity of the oxygen sensor, prolyl hydroxylase, and oxygen reserve, in the developing heart. We tested the role of hypoxia-dependent responses in heart development by targeted inactivation of Hif-1α. METHODS AND RESULTS: ODD-Luciferase activity was 14-fold higher in mouse embryonic day 10.5 (E10.5) versus adult heart and liver tissue lysates. ODD-Luc activity decreased in 2 stages, the first corresponding with the formation of a functional cardiovascular system for oxygen delivery at E15.5, and the second after birth consistent with complete oxygenation of the blood and tissues. Reduction of maternal inspired oxygen to 8% for 4 hours caused minimal induction of luciferase activity in the maternal tissues but robust induction in the embryonic tissues in proportion to the basal activity, indicating a lack of oxygen reserve, and corresponding induction of a hypoxia-dependent gene program. Bioluminescent imaging of intact embryos demonstrated highest activity in the outflow portion of the E13.5 heart. Hif-1α inactivation or prolonged hypoxia caused outflow and septation defects only when targeted to this specific developmental window. CONCLUSIONS: Low oxygen concentrations and lack of oxygen reserve during a critical phase of heart organogenesis may provide a basis for vulnerability to the development of common septation and conotruncal heart defects.

Prostaglandin E receptor EP4 is a therapeutic target in breast cancer cells with stem-like properties.

The cyclooxygenase pathway is strongly implicated in breast cancer progression but the role of this pathway in the biology of breast cancer stem/progenitor cells has not been defined. Recent attention has focused on targeting the cyclooxygenase 2 (COX-2) pathway downstream of the COX-2 enzyme by blocking the activities of individual prostaglandin E (EP) receptors. Prostaglandin E receptor 4 (EP4) is widely expressed in primary invasive ductal carcinomas of the breast and antagonizing this receptor with small molecule inhibitors or shRNA directed to EP4 inhibits metastatic potential in both syngeneic and xenograft models. Breast cancer stem/progenitor cells are defined as a subpopulation of cells that drive tumor growth, metastasis, treatment resistance, and relapse. Mammosphere-forming breast cancer cells of human (MDA-MB-231, SKBR3) or murine (66.1, 410.4) origin of basal-type, Her-2 phenotype and/or with heightened metastatic capacity upregulate expression of both EP4 and COX-2 and are more tumorigenic compared to the bulk population. In contrast, luminal-type or non-metastatic counterparts (MCF7, 410, 67) do not increase COX-2 and EP4 expression in mammosphere culture. Treatment of mammosphere-forming cells with EP4 inhibitors (RQ-15986, AH23848, Frondoside A) or EP4 gene silencing, but not with a COX inhibitor (Indomethacin) reduces both mammosphere-forming capacity and the expression of phenotypic markers (CD44(hi)/CD24(low), aldehyde dehydrogenase) of breast cancer stem cells. Finally, an orally delivered EP4 antagonist (RQ-08) reduces the tumor-initiating capacity and markedly inhibits both the size of tumors arising from transplantation of mammosphere-forming cells and phenotypic markers of stem cells in vivo. These studies support the continued investigation of EP4 as a potential therapeutic target and provide new insight regarding the role of EP4 in supporting a breast cancer stem cell/tumor-initiating phenotype.

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.