Radiation therapy affects YAP expression and intracellular localization by modulating lamin A/C levels in breast cancer

The microenvironment of breast cancer actively participates in tumorigenesis and cancer progression. The changes observed in the architecture of the extracellular matrix initiate an oncogene-mediated cell reprogramming, that leads to a massive triggering of YAP nuclear entry, and, therefore, to cancer cell proliferation, invasion and probably to increased radiation-resistance. However, it is not yet fully understood how radiotherapy regulates the expression and subcellular localization of YAP in breast cancer cells experiencing different microenvironmental stiffnesses. To elucidate the role of extracellular matrix stiffness and ionizing radiations on YAP regulation, we explored the behaviour of two different mammary cell lines, a normal epithelial cell line (MCF10A) and a highly aggressive and invasive adenocarcinoma cell line (MDA-MB-231) interacting with polyacrylamide substrates mimicking the mechanics of both normal and tumour tissues (~1 and ~13 kPa). We report that X-ray radiation affected in a significant way the levels of YAP expression, density, and localization in both cell lines. After 24 h, MCF10A and MDA-MB231 increased the expression level of YAP in both nucleus and cytoplasm in a dose dependent manner and particularly on the stiffer substrates. After 72 h, MCF10A reduced mostly the YAP expression in the cytoplasm, whereas it remained high in the nucleus of cells on stiffer substrates. Tumour cells continued to exhibit higher levels of YAP expression, especially in the cytoplasmic compartment, as indicated by the reduction of nuclear/ cytoplasmic ratio of total YAP. Then, we investigated the existence of a correlation between YAP localization and the expression of the nuclear envelope protein lamin A/C, considering its key role in modulating nuclear deformability and changes in YAP shuttling phenomena. As supposed, we found that the effects of radiation on YAP nucleus/cytoplasmic expression ratio, increasing in healthy cells and decreasing in tumour ones, were accompanied by lower and higher lamin A/C levels in MCF10A and MDAMB-231 cells, respectively. These findings point to obtain a deeper knowledge of the role of the extracellular matrix and the effects of X-rays on YAP and lamin A/C expression that can be used in the design of doses and timing of radiation therapy

X-rays affect cytoskeleton assembly and nanoparticle uptake: Preliminary results

Alterations of the cytoskeleton are commonly associated with tumor genesis and cancer progression. For this reason, the characterization of cytoskeletonassociated functions and properties is important to optimize the outcomes to classical and more recent therapeutic approaches, such as chemotherapy, radiotherapy and cancer nanomedicine. In such context, this work investigated the synergy between cancer nanomedicine and radiotherapy. In particular, the effects over time (24 and 48h) of two different doses of X-rays (2 and 10Gy) on spreading area, morphological parameters and the internalization mechanism of carboxylated nanoparticles in mammary epithelial cells and mammary adenocarcinoma cells were investigated

Investigation of Biophysical Migration Parameters for Normal Tissue and Metastatic Cancer Cells After Radiotherapy Treatment

A large body of literature has demonstrated that the mechanical properties of microenvironment have a key role in regulating cancer cell adhesion, motility, and invasion. In this work, we have introduced two additional parameters, named cell trajectory extension and area traveled by cell, to describe the tendency of normal tissue and metastatic cancer cells to move in a directional way when they interact with physio-pathological substrates, characterized by stiffnesses of 1–13 kPa, before and after treatment with 2 doses of X-rays (2 and 10 Gy). We interpreted these data by evaluating also the impact of substrate stiffness on 2 morphological parameters which indicate not only the state of cell adhesion, but also cell polarization, prerequisite to directional movement, and the formation of protrusions over cell perimeters. We believe that a so wide analysis can give an efficient and easily readable overview of effects of radiation therapy on cell-ECM crosstalk when used as therapeutic agent

Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)–Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK–Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell’s environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies

Evaluation of material mechanical properties influence on single cell mechanics

Abstract Mechanobiology research has shown that mechanical signals influence a wide spectrum of cellular events, including cell proliferation, differentiation, gene expression, protein production and their alterations. The objective of this projects is to elucidate the role of two mechanical factors, matrix stiffness and externally applied forces, in the organization and contractile activity of the cytoskeleton and distribution of intracellular forces. Indeed, localized concentration of cytoskeletal tensions at focal adhesions, the structures that link cells to their surrounding extracellular matrix, is the major mediator of mechanical signaling. Therefore, in the first phase of project we have studied how matrix stiffness in coordination with surface functionalization can regulate shape and the structural organization of integrated system constituted by actin network and integrin-mediated adhesion of fibroblasts. Then, we have investigated if there is a direct correlation between ECM stiffness and intracellular mechanics, measuring mechanical properties by particle tracking technique. A mechanical model has been developed to support experimental results and explain the relation that exists between matrix rigidity, focal adhesion sites dimension, cytoskeleton structure and intracellular mechanics. In the second part of project, we have focused attention on how integration of externally applied mechanical forces from focal adhesions over the entire cell body affects fibroblast responses to its mechanical environments both in 2D and in 3D matrix. In conclusion, we have observed that both matrix stiffness and external mechanical stress represent important stimuli to enhance cell stiffness and contractility of fibroblasts through cytoskeleton structuration, indicating that mechanics plays a critical role in cell biology. This consideration provides a solid foundation and rationale for use of mechanics to improve human health by designing appropriate equipment/instruments, exercise protocols, and rehabilitation regimens

Mechanosensing of substrate stiffness regulates focal adhesions dynamics in cell

Cell mechanical recognition of extracellular matrix determines the cell activities and functions. Focal adhesions are part of the cell mechanosensing machinery and, operating at the very dynamic interface between cell and extracellular matrix, can operate this recognition and trigger conformational, functional and behavioral modification of the cell. To investigate how the dynamic of assembly and disassembly of focal adhesion are influenced by the substrate mechanics we developed a novel procedure. The analysis consists of the over time tracking of focal adhesion structures in a stable cell line of NIH/3T3 expressing fluorescent pmKate2-paxillin. From collected signals and by their autocorrelation we evaluated the average lifetime and assembly rate of focal adhesion as function of substrate stiffness. Further, by signals cross-correlation we obtained information about the mechanical nature of cytoskeleton and its network. This quantitative approach to focal adhesion dynamics characterization was presented in this study as an investigation tool for cell mechanobiology

ECM mechanoregulation in malignant pleural mesothelioma

Malignant pleural mesothelioma is a relatively rare, but devastating tumor, because of the difficulties in providing early diagnosis and effective treatments with conventional chemo- and radiotherapies. Patients usually present pleural effusions that can be used for diagnostic purposes by cytological analysis. This effusion cytology may take weeks or months to establish and has a limited sensitivity (30%–60%). Then, it is becoming increasingly urgent to develop alternative investigative methods to support the diagnosis of mesothelioma at an early stage when this cancer can be treated successfully. To this purpose, mechanobiology provides novel perspectives into the study of tumor onset and progression and new diagnostic tools for the mechanical characterization of tumor tissues. Here, we report a mechanical and biophysical characterization of malignant pleural mesothelioma cells as additional support to the diagnosis of pleural effusions. In particular, we examined a normal mesothelial cell line (Met5A) and two epithelioid mesothelioma cell lines (REN and MPP89), investigating how malignant transformation can influence cellular function like proliferation, cell migration, and cell spreading area with respect to the normal ones. These alterations also correlated with variations in cytoskeletal mechanical properties that, in turn, were measured on substrates mimicking the stiffness of patho-physiological ECM

Crosstalk between focal adhesions and material mechanical properties governs cell mechanics and functions

Mechanical properties of materials strongly influence cell fate and functions. Focal adhesions are involved in the extremely important processes of mechanosensing and mechanotransduction. To address the relationship between the mechanical properties of cell substrates, focal adhesion/cytoskeleton assembly and cell functions, we investigated the behavior of NIH/3T3 cells over a wide range of stiffness (3-1000 kPa) using two of the most common synthetic polymers for cell cultures: polyacrylamide and polydimethylsiloxane. An overlapping stiffness region was created between them to compare focal adhesion characteristics and cell functions, taking into account their different time-dependent behavior. Indeed, from a rheological point of view, polyacrylamide behaves like a strong gel (elastically), whereas polydimethylsiloxane like a viscoelastic solid. First, focal adhesion characteristics and dynamics were addressed in terms of material stiffness, then cell spreading area, migration rate and cell mechanical properties were correlated with focal adhesion size and assembly. Focal adhesion size was found to increase in the whole range of stiffness and to be in agreement in the overlapping rigidity region for the investigated materials. Cell mechanics directly correlated with focal adhesion lengths, whereas migration rate followed an inverse correlation. Cell spreading correlated with the substrate stiffness on polyacrylamide hydrogel, while no specific trend was found on polydimethylsiloxane. Substrate mechanics can be considered as a key physical cue that regulates focal adhesion assembly, which in turn governs important cellular properties and functions