Stephen J. P. Pratt

The tumor microenvironment and healing wounds both contain extremely high concentrations of adenosine triphosphate (ATP) compared to normal tissue. The P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here, we examine ATP-dependent signaling in breast epithelial cells and how it is altered in metastatic breast cancer. Using rapid imaging techniques, we show how ATP-activated P2Y2 signaling causes an increase in intracellular Ca²⁺ in non-tumorigenic breast epithelial cells, approximately 3-fold higher than their tumorigenic and metastatic counterparts. The non-tumorigenic cells respond to increased Ca²⁺ with actin polymerization and localization to the cell edges after phalloidin staining, while the metastatic cells remain unaffected. The increase in intracellular Ca²⁺ after ATP stimulation was blunted to control levels using a P2Y2 antagonist, which also prevented actin mobilization and significantly increased cell dissemination from spheroids in non-tumorigenic cells. Furthermore, the lack of Ca²⁺ changes and actin mobilization in metastatic breast cancer cells could be due to the reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates the rapid changes that occur after elevated intracellular Ca²⁺ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.

Mechanobiology is a rapidly advancing field, with growing evidence that mechanical signaling plays key roles in health and disease. To accelerate mechanobiology-based drug discovery, novel in vitro systems are needed that enable mechanical perturbation of cells in a format amenable to high throughput screening. Here, both a mechanical stretch device and 192-well silicone flexible linear stretch plate were designed and fabricated to meet high throughput technology needs for cell stretch-based applications. To demonstrate the utility of the stretch plate in automation and screening, cell dispensing, liquid handling, high content imaging, and high throughput sequencing platforms were employed. Using this system, an assay was developed as a biological validation and proof-of-concept readout for screening. A mechano-transcriptional stretch response was characterized using focused gene expression profiling measured by RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing. Using articular chondrocytes, a gene expression signature containing stretch responsive genes relevant to cartilage homeostasis and disease was identified. The possibility for integration of other stretch sensitive cell types (e.g., cardiovascular, airway, bladder, gut, and musculoskeletal), in combination with alternative phenotypic readouts (e.g., protein expression, proliferation, or spatial alignment), broadens the scope of high throughput stretch and allows for wider adoption by the research community. This high throughput mechanical stress device fills an unmet need in phenotypic screening technology to support drug discovery in mechanobiology-based disease areas.

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.

Calcium is a versatile element that participates in cell signaling for a wide range of cell processes such as death, cell cycle, division, migration, invasion, metabolism, differentiation, autophagy, transcription, and others. Specificity of calcium in each of these processes is achieved through modulation of intracellular calcium concentrations by changing the characteristics (amplitude/frequency modulation) or location (spatial modulation) of the signal. Breast cancer utilizes calcium signaling as an advantage for survival and progression. This review integrates evidence showing that increases in expression of calcium channels, GPCRs, pumps, effectors, and enzymes, as well as resulting intracellular calcium signals, lead to high calcium and/or an elevated calcium- mobilizing capacity necessary for malignant functions such as migratory, invasive, proliferative, tumorigenic, or metastatic capacities.

Significance: In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities.

Mechanotransduction is the interpretation of physical cues by cells through mechanosensation mechanisms that elegantly translate mechanical stimuli into biochemical signaling pathways. While mechanical stress and their resulting cellular responses occur in normal physiologic contexts, there are a variety of cancer-associated physical cues present in the tumor microenvironment that are pathological in breast cancer. Mechanistic in vitro data and in vivo evidence currently support three mechanical stressors as mechanical modifiers in breast cancer that will be the focus of this review: stiffness, interstitial fluid pressure, and solid stress. Increases in stiffness, interstitial fluid pressure, and solid stress are thought to promote malignant phenotypes in normal breast epithelial cells, as well as exacerbate malignant phenotypes in breast cancer cells.

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

The skeletal muscle Ca2+ release channel, also known as ryanodine receptor type 1 (RyR1), is the largest ion channel protein known and is crucial for effective skeletal muscle contractile activation. RyR1 function is controlled by Cav1.1, a voltage gated Ca2+ channel that works mainly as a voltage sensor for RyR1 activity during skeletal muscle contraction and is also fine-tuned by Ca2+, several intracellular compounds (e.g., ATP), and modulatory proteins (e.g., calmodulin). Dominant and recessive mutations in RyR1, as well as acquired channel alterations, are the underlying cause of various skeletal muscle diseases. The aim of this mini review is to summarize several current aspects of RyR1 function, structure, regulation, and to describe the most common diseases caused by hereditary or acquired RyR1 malfunction.