Focal Adhesion Complexes Research Articles

Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells.

Glioblastomas are the most common and aggressive primary brain tumors in adults, and patients with glioblastoma have a median survival of 15 months. Some alternative therapies, such as Src family kinase inhibitors, have failed presumably because other signaling pathways compensate for their effects. In the last ten years, it has been proven that sex hormones such as progesterone (P4) can induce growth, migration, and invasion of glioblastoma cells through its intracellular progesterone receptor (PR), which is mostly known for its role as a transcription factor, but it can also induce non-genomic actions. These non-classic actions are, in part, a consequence of its interaction with cSrc, which plays a significant role in the progression of glioblastomas. We studied the relation between PR and cSrc, and its effects in human glioblastoma cells. Our results showed that P4 and R5020 (specific PR agonist) activated cSrc protein since both progestins increased the p-cSrc (Y416)/cSrc ratio in U251 and U87 human glioblastoma derived cell lines. When siRNA against the PR gene was used, the activation of cSrc by P4 was abolished. The co-immunoprecipitation assay showed that cSrc and PR interact in U251 cells. P4 treatment also promoted the increase in the p-Fak (Y397) (Y576/577)/Fak and the decrease in p-Paxillin (Y118)/Paxillin ratio, which are significant components of the focal adhesion complex and essential for migration and invasion processes. A siRNA against cSrc gene blocked the increase in the p-Fak (Y576/Y577)/Fak ratio and the migration induced by P4, but not the decrease in p-Paxillin (Y118)/Paxillin ratio. We analyzed the potential role of cSrc over PR phosphorylation in three databases, and one putative tyrosine residue in the amino acid 87 of PR was found. Our results showed that P4 induces the activation of cSrc protein through its PR. The latter and cSrc could interact in a bidirectional mode for regulating the activity of proteins involved in migration and invasion of glioblastomas.

C-Jun N-terminal kinase (JNK) pathway activation is essential for dental papilla cells polarization.

During tooth development, dental papilla cells differentiate into odontoblasts with polarized morphology and cell function. Our previous study indicated that the C-Jun N-terminal kinase (JNK) pathway regulates human dental papilla cell adhesion, migration, and formation of focal adhesion complexes. The aim of this study was to further examine the role of the JNK pathway in dental papilla cell polarity formation. Histological staining, qPCR, and Western Blot suggested the activation of JNK signaling in polarized mouse dental papilla tissue. After performing an in vitro tooth germ organ culture and cell culture, we found that JNK inhibitor SP600125 postponed tooth germ development and reduced the polarization, migration and differentiation of mouse dental papilla cells (mDPCs). Next, we screened up-regulated polarity-related genes during dental papilla development and mDPCs or A11 differentiation. We found that Prickle3, Golga2, Golga5, and RhoA were all up-regulated, which is consistent with JNK signaling activation. Further, constitutively active RhoA mutant (RhoA Q63L) partly rescued the inhibition of SP600125 on cell differentiation and polarity formation of mDPCs. To sum up, this study suggests that JNK signaling has a positive role in the formation of dental papilla cell polarization.

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Cell migration is an essential cellular process that requires coordination of cytoskeletal dynamics, reorganization, and signal transduction. The actin cytoskeleton is central in maintaining the cellular structure as well as regulating the mechanisms of cell motility. Glycosylation, particularly sialylation of cell surface proteins like integrins, regulates signal transduction from the extracellular matrix to the cytoskeletal network. The activation of integrin by extracellular cues leads to recruitment of different focal adhesion complex proteins (Src, FAK, paxillin, etc.) and activates the signal including Rho GTPases for the regulation of actin assembly and disassembly. During cell migration, the assembly and disassembly of actin filament provides the essential force for the cell to move. Abnormal sialylation can lead to actin signaling dysfunction leading to aberrant cell migration, one of the main characteristics of cancer and myopathies. In the present study, we have reported altered F-actin to G-actin ratios in GNE mutated cells. These cells exhibit pathologically relevant mutations of GNE (UDP N-acetylneuraminic 2-epimerase/N-acetylmannosamine kinase), a key sialic acid biosynthetic enzyme. It was found that GNE neither affects the actin polymerization nor binds directly to actin. However, mutation in GNE resulted in increased binding of α-actinin to actin filaments. Further, through confocal imaging, GNE was found to be localized in focal adhesion complex along with paxillin. We further elucidated that mutation in GNE resulted in upregulation of RhoA protein and Cofilin activity is downregulated, which could be rescued with Rhosin and chlorogenic acid, respectively. Lastly, mutant in GNE reduced cell migration as implicated from wound healing assay. Our study indicates that molecules altering Cofilin function could significantly revert the cell migration defect due to GNE mutation in sialic acid-deficient cells. We propose cytoskeletal proteins to be alternate drug targets for disorders associated with GNE such as GNE myopathy.

Molecular Basis of LH Action on Breast Cancer Cell Migration and Invasion via Kinase and Scaffold Proteins.

Breast cancer (BC) is a major public health problem affecting women worldwide. Approximately 80% of diagnosed cases are hormone-dependent breast cancers. These hormones are known to stimulate tumor development and progression. In this setting, tentative evidence suggests that luteinizing hormone (LH) may also play a role in tumors. In BC cells that express functional LH receptors (LHR), this hormone regulates cell migration and invasion by controlling several kinases that activate actin cytoskeletal proteins. In this article, we show that LH induces phosphorylation of paxillin and its translocation toward the plasmatic membrane, where focal adhesion complexes are assembled. This process is triggered via a rapid extra-gonadal LHR signaling to Src/FAK/paxillin, which results in the phosphorylation/activation of the nucleation promoter factors cortactin and N-WASP. As a consequence, Arp2/3 complexes induce actin polymerization, essential to promote cell adhesion, migration, and invasion, thus enhancing metastatic spread of tumoral cells. Our findings provide relevant information about how gonadotrophins exert their action in BC. This information helps us understand the extragonadal effects of LH on BC metastasis. It may provide new perspectives for therapeutic treatment, especially for women with high serum levels of gonadotrophins.

Enhanced Sampling Molecular Dynamics to Determine Force Response in Peptide Tension Sensors

Mechanical forces are prevalent in diverse cellular processes, such as cell division, motility and adhesion. “Mechanoresponsive” proteins are present which respond to these applied forces by changing their structure to perform some function. Scientists have developed Tension Sensing modules to determine the size and location of forces associated with Mechanoresponsive protein assemblies such as Focal Adhesions complexes. However, it is not well understood why certain peptides are better suited to be tension sensors nor in general how to predict mechanical properties from sequence. As a consequence, it is not clear why different tensions sensors are optimally responsive in different force regimes. Current experimental approaches to study piconewton (pN) forces such as single force microscopy are often difficult to implement to study changes under such small forces. However, Molecular Dynamics (MD) and Enhanced sampling techniques can be used to study the effect of applied forces on peptides at the molecular level. Here we will present our work to develop methodologies that can rapidly predict the force-extension behavior of small peptides, and show our recent developments that will allow us to predict the behavior of full-length tension sensing peptide modules.

Heregulin-induced cell migration is prevented by trastuzumab and trastuzumab-emtansine in HER2+ breast cancer.

Heregulin (HRG) signaling has been implicated in the development of an aggressive phenotype in breast cancer (BC) cells, and HER2 overexpression has been associated with a worse prognosis in BC patients. Nevertheless, the molecular mechanisms through which HRG affects the efficiency of anti-HER2 therapies such as trastuzumab (Tz) and trastuzumab-emtansine (T-DM1) are currently unknown. In the present study, we evaluate the molecular action of HRG toward fundamental scaffold proteins and several kinases in the signal transduction pathways triggered via HER2/HER3, which integrate precise and sequential steps to promote changes in cell morphology to impulse BC cell migration. In addition, we evaluate the effectiveness of Tz and T-DM1 on the control of key proteins involved in BC cell motility, since the acquisition of a migratory phenotype is essential to promote invasion and metastasis. We show that HRG induces actin cytoskeleton reorganization and focal adhesion complex formation, and promotes actin nucleation in BT-474 BC cells. This signaling is triggered by HER2/HER3 to c-Src, FAK and paxillin. When paxillin is phosphorylated, it recruits PAK1, which then phosphorylates cortactin. In parallel, paxillin signals to N-WASP, and both signalings regulate Arp2/3 complex, leading to the local reorganization of actin fibers. Our findings reveal an original mechanism by which HRG increases HER2+ BC cell motility, and show that the latter can be abolished by Tz and T-DM1 treatments. These results provide evidence for the molecular mechanisms involved in cell motility and drug resistance. They will be useful to develop new and more specific therapeutic schemes that interfere with the progression and metastasis of HER2+ BC.

Confirming whether KLHL23 deficiency potentiates migration in urothelial carcinoma.

Epithelial-mesenchymal transition (EMT) is associated with malignant tumors. In a previous study, we found that KLHL23 is a tumor suppressor gene that inhibits EMT and cancer dissemination. However, the correlation between its expression and cancer progression in urothelial carcinoma (UC) remains unknown. This study showed that the deficiency of KLHL23 in the invasive leading cancer cells is important for improving cell migration in UC. Currently, little is known about the underlying mechanisms of KLHL23-mediated cytoskeleton remodeling in the metastatic leading cells of tumors. Our findings showed that silencing of KLHL23 promotes cell migration in UC by regulating the translocation of focal adhesion proteins. Lack of KLHL23 causes abnormal formation of lamellipodia and increases the EMT phenotype and migration. Wound healing assay revealed that KLHL23 potentiates the actin bundles and intracellular focal adhesion protein formation in the invasive leading cells. Knockdown of KLHL23 abolishes the formation of actin stress fibers and translocalizes vinculin to the perimembrane, which enhances the mobility of cancer cells. To elucidate the mechanism, we found that during migration, KLHL23 appears in the leading cells in large numbers and binds to the actin stress fibers. A large amount of vinculin accumulated at both ends of the KLHL23/actin fibers, indicating an increase in cell anchorage. Thus, KLHL23 might play a critical role in enhancing actin fibers and promoting focal adhesion complex formation in the invasive leading cells. Analysis of the overall survival revealed that low KLHL23 is associated with poor survival in patients with bladder UC, indicating its clinical significance. We hypothesize that KLHL23 is involved in the formation of actin stress fibers and focal adhesion complexes in the invasive leading cells and may be associated with EMT progression and prognosis in UC patients.

Rab11-mediated focal adhesion turnover in sarcoma cell migration.

Focal adhesion (FA) turnover has been demonstrated to play an important role in cell migration; however, the mechanism of FA turnover is complicated and requires further investigation. In this study, Rab11, which is involved in endosome recycling, was examined in terms of a direct regulatory function in FA formation during cell migration. Wild-type and dominant negative (DN) Rab11 or shRab11 were transfected into human HT1080 fibrosarcoma cells; the cell motility and migration abilities were determined, and localization of Rab11 and FA molecules was monitored by confocal microscopy. The results showed that Rab11 deficiency or the DN form inhibited sarcoma cell migration. Rab11 was also found to be co-localized with recycled β1 integrin and affected FA formation. We further employed immunofluorescence and immunoprecipitation to examine the physical interaction between Rab11 and focal adhesion kinase (FAK), and the results suggested that Rab11 affected cell migration by regulating FAK recycling to aid formation of an FA complex on the cell membrane.

SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins.

Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Cx43 and the Actin Cytoskeleton: Novel Roles and Implications for Cell-Cell Junction-Based Barrier Function Regulation.

Barrier function is a vital homeostatic mechanism employed by epithelial and endothelial tissue. Diseases across a wide range of tissue types involve dynamic changes in transcellular junctional complexes and the actin cytoskeleton in the regulation of substance exchange across tissue compartments. In this review, we focus on the contribution of the gap junction protein, Cx43, to the biophysical and biochemical regulation of barrier function. First, we introduce the structure and canonical channel-dependent functions of Cx43. Second, we define barrier function and examine the key molecular structures fundamental to its regulation. Third, we survey the literature on the channel-dependent roles of connexins in barrier function, with an emphasis on the role of Cx43 and the actin cytoskeleton. Lastly, we discuss findings on the channel-independent roles of Cx43 in its associations with the actin cytoskeleton and focal adhesion structures highlighted by PI3K signaling, in the potential modulation of cellular barriers. Mounting evidence of crosstalk between connexins, the cytoskeleton, focal adhesion complexes, and junctional structures has led to a growing appreciation of how barrier-modulating mechanisms may work together to effect solute and cellular flux across tissue boundaries. This new understanding could translate into improved therapeutic outcomes in the treatment of barrier-associated diseases.

Biallelic mutations in LAMA5 disrupts a skeletal noncanonical focal adhesion pathway and produces a distinct bent bone dysplasia.

BackgroundBeyond its structural role in the skeleton, the extracellular matrix (ECM), particularly basement membrane proteins, facilitates communication with intracellular signaling pathways and cell to cell interactions to control differentiation, proliferation, migration and survival. Alterations in extracellular proteins cause a number of skeletal disorders, yet the consequences of an abnormal ECM on cellular communication remains less well understoodMethodsClinical and radiographic examinations defined the phenotype in this unappreciated bent bone skeletal disorder. Exome analysis identified the genetic alteration, confirmed by Sanger sequencing. Quantitative PCR, western blot analyses, immunohistochemistry, luciferase assay for WNT signaling were employed to determine RNA, proteins levels and localization, and dissect out the underlying cell signaling abnormalities. Migration and wound healing assays examined cell migration properties.FindingsThis bent bone dysplasia resulted from biallelic mutations in LAMA5, the gene encoding the alpha-5 laminin basement membrane protein. This finding uncovered a mechanism of disease driven by ECM-cell interactions between alpha-5-containing laminins, and integrin-mediated focal adhesion signaling, particularly in cartilage. Loss of LAMA5 altered β1 integrin signaling through the non-canonical kinase PYK2 and the skeletal enriched SRC kinase, FYN. Loss of LAMA5 negatively impacted the actin cytoskeleton, vinculin localization, and WNT signaling.InterpretationThis newly described mechanism revealed a LAMA5-β1 Integrin-PYK2-FYN focal adhesion complex that regulates skeletogenesis, impacted WNT signaling and, when dysregulated, produced a distinct skeletal disorder.FundingSupported by NIH awards R01 AR066124, R01 DE019567, R01 HD070394, and U54HG006493, and Czech Republic grants INTER-ACTION LTAUSA19030, V18-08-00567 and GA19-20123S.

Focal Adhesion Isolation Assay Using ECM-Coated Magnetic Beads.

Focal adhesions are force sensitive structures that dynamically alter their composition, protein-protein interactions, and signaling in response to external mechanical stimuli. These dynamic changes are critical for focal adhesion function and are required for cellular mechanosensing. Here, we describe a simple protocol that allows for isolation of the focal adhesion complex from adherent cells in culture in response to different mechanical stimuli applied at adhesion sites. By combining this assay with approaches such as proteomics or western blot analysis, one can study the force-dependent changes in focal adhesion composition, protein-protein interactions and signaling.

Chitosan-GPTMS-Silica Hybrid Mesoporous Aerogels for Bone Tissue Engineering.

This study introduces a new synthesis route for obtaining homogeneous chitosan (CS)-silica hybrid aerogels with CS contents up to 10 wt%, using 3-glycidoxypropyl trimethoxysilane (GPTMS) as coupling agent, for tissue engineering applications. Aerogels were obtained using the sol-gel process followed by CO2 supercritical drying, resulting in samples with bulk densities ranging from 0.17 g/cm3 to 0.38 g/cm3. The textural analysis by N2-physisorption revealed an interconnected mesopore network with decreasing specific surface areas (1230–700 m2/g) and pore sizes (11.1–8.7 nm) by increasing GPTMS content (2–4 molar ratio GPTMS:CS monomer). In addition, samples exhibited extremely fast swelling by spontaneous capillary imbibition in PBS solution, presenting swelling capacities from 1.75 to 3.75. The formation of a covalent crosslinked hybrid structure was suggested by FTIR and confirmed by an increase of four hundred fold or more in the compressive strength up to 96 MPa. Instead, samples synthesized without GPTMS fractured at only 0.10–0.26 MPa, revealing a week structure consisted in interpenetrated polymer networks. The aerogels presented bioactivity in simulated body fluid (SBF), as confirmed by the in vitro formation of hydroxyapatite (HAp) layer with crystal size of approximately 2 µm size in diameter. In vitro studies revealed also non cytotoxic effect on HOB® osteoblasts and also a mechanosensitive response. Additionally, control cells grown on glass developed scarce or no stress fibers, while cells grown on hybrid samples showed a significant (p < 0.05) increase in well-developed stress fibers and mature focal adhesion complexes.

The hidden role of paxillin: localization to nucleus promotes tumor angiogenesis.

Paxillin (PXN), a key component of the focal adhesion complex, has been associated with cancer progression, but the underlying mechanisms are poorly understood. The purpose of this study was to elucidate mechanisms by which PXN affects cancer growth and progression, which we addressed using cancer patient data, cell lines, and orthotopic mouse models. We demonstrated a previously unrecognized mechanism whereby nuclear PXN enhances angiogenesis by transcriptionally regulating SRC expression. SRC, in turn, increases PLAT expression through NF-ĸB activation; PLAT promotes angiogenesis via LRP1 in endothelial cells. PXN silencing in ovarian cancer mouse models reduced angiogenesis, tumor growth, and metastasis. These findings provide a new understanding of the role of PXN in regulating tumor angiogenesis and growth.

Lamellipodia-like protrusions and focal adhesions contribute to collective cell migration in zebrafish

Collective cell migration is a process where cohorts of cells exhibit coordinated migratory behavior. During individual and collective cellular migration, cells must extend protrusions to interact with the extracellular environment, sense chemotactic cues, and act as points of attachment. The mechanisms and regulators of protrusive behavior have been widely studied in individually migrating cells; however, how this behavior is regulated throughout collectives is not well understood. To address this, we used the zebrafish posterior lateral line primordium (pLLP) as a model. The pLLP is a cluster of ~150 ​cells that migrates along the zebrafish trunk, depositing groups of cells that will become sensory organs. To define protrusive behavior, we performed mosaic analysis to sparsely label pLLP cells with a transgene marking filamentous actin. This approach revealed an abundance of brush-like protrusions throughout the pLLP that orient in the direction of migration. Formation of these protrusions depends on the Arp2/3 complex, a regulator of dendritic actin. This argues that these brush-like protrusions are an in vivo example of lamellipodia. Mosaic analysis demonstrated that these lamellipodia-like protrusions are located in a close proximity to the overlying skin. Immunostaining revealed an abundance of focal adhesion complexes surrounding the pLLP. Disruption of these complexes specifically in pLLP cells led to impaired pLLP migration. Finally, we show that Erk signaling, a known regulator of focal adhesions, is required for proper formation of lamellipodia-like protrusions and pLLP migration. Altogether, our results suggest a model where the coordinated dynamics of lamellipodia-like protrusions, making contact with either the overlying skin or the extracellular matrix through focal adhesions, promotes migration of pLLP cells.

Focal adhesion ribonucleoprotein complex proteins are major humoral cancer antigens and targets in autoimmune diseases

Despite the accumulating evidences of the significance of humoral cancer immunity, its molecular mechanisms have largely remained elusive. Here we show that B-cell repertoire sequencing of 102 clinical gastric cancers and molecular biological analyses unexpectedly reveal that the major humoral cancer antigens are not case-specific neo-antigens but are rather commonly identified as ribonucleoproteins (RNPs) in the focal adhesion complex. These common antigens are shared as autoantigens with multiple autoimmune diseases, suggesting a direct molecular link between cancer- and auto-immunity on the focal adhesion RNP complex. This complex is partially exposed to the outside of cancer cell surfaces, which directly evokes humoral immunity and enables functional bindings of antibodies to cancer cell surfaces in physiological conditions. These findings shed light on humoral cancer immunity in that it commonly targets cellular components fundamental for cytoskeletal integrity and cell movement, pointing to a novel modality of immunotherapy using humoral immunological reactions to cancers.

Mitigative Effect of Sipjeondaebo-tang on RhoA Activation in Cold-Exposed Vascular Cells

Objective To investigate the vasodilatory effect of Sipjeondaebo-tang by inhibiting RhoA activity in vascular cells during cold exposure. Methods Human vascular endothelial cells and pericytes were pretreated with Sipjeondaebo-tang for 30 min, followed by incubation at 37 °C (control) or 25 °C (cold exposure) for 30 min. Activation of endothelin-1-mediated RhoA in pericytes was assessed by pretreating the cells with Sipjeondaebo-tang for 30 min, followed by incubation with endothelin-1 at 37 °C for 30 min. Western blotting was performed to measure the expression of active RhoA. Endothelin-1 and nitric oxide release from endothelial cells was examined with enzyme-linked immunosorbent assay kits. The formation of stress fibers and focal adhesion complexes was analyzed by immunocytochemistry. Results Cold treatment activated RhoA in both pericytes and vascular endothelial cells, whereas Sipjeondaebo-tang treatment inhibited this activation. Sipjeondaebo-tang treatment also reversed the cold-mediated production of endothelin-1 and nitric oxide. Cold exposure promoted the formation of stress fibers and focal adhesion complexes by increasing the expression of phospho-focal adhesion complex kinase, whereas Sipjeondaebo-tang treatment suppressed this response. Conclusions These findings suggested that Sipjeondaebo-tang inhibits cold-induced RhoA activation and its related pathway components, including endothelin-1 and nitric oxide, in vascular cells. Therefore, Sipjeondaebo-tang could be beneficial for the treatment of Raynaud’s phenomenon. Keywords: Raynaud’s phenomenon, cold, rhoA, endothelin-1, nitric oxide, Sipjeondaebo-tang

A Non-genetic Mechanism Involving the Integrin β4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

SummaryTumor heterogeneity and cisplatin resistance are major causes of tumor relapse and poor survival. Here, we show that in lung cancer, interaction between paxillin (PXN) and integrin β4 (ITGB4), components of the focal adhesion (FA) complex, contributes to cisplatin resistance. Knocking down PXN and ITGB4 attenuated cell growth and improved cisplatin sensitivity, both in 2D and 3D cultures. PXN and ITGB4 independently regulated expression of several genes. In addition, they also regulated expression of common genes including USP1 and VDAC1, which are required for maintaining genomic stability and mitochondrial function, respectively. Mathematical modeling suggested that bistability could lead to stochastic phenotypic switching between cisplatin-sensitive and resistant states in these cells. Consistently, purified subpopulations of sensitive and resistant cells re-created the mixed parental population when cultured separately. Altogether, these data point to an unexpected role of the FA complex in cisplatin resistance and highlight a novel non-genetic mechanism.

Abstract 6329: Carfilzomib: A potential drug against cisplatin-resistant lung adenocarcinoma

Abstract Recent advances in the treatment of lung cancer, such as small molecule inhibitors and immunotherapy, have transformed care. However, platinum-based chemotherapy, such as cisplatin, is the recommended treatment option for those that do not express markers that respond to targeted therapy. In most cases, prolonged exposure to cisplatin eventually renders cancer cells resistant to therapy and patients progress on this line of care. Our study has shown that integrin beta 4 (ITGB4) and paxillin (PXN) in the focal adhesion complex are highly expressed in lung adenocarcinoma (LUAD) and their interaction plays an important role in mediating cisplatin resistance. Disrupting this axis with an FDA-approved compound can potentially alleviate cisplatin resistance in non-small cell lung cancer. Upon screening 10 LUAD cell lines, we determined one KRAS mutant (H2009) and one KRAS wild-type cell line (H1993) with highest resistance to cisplatin using a cell viability assay. Proximity ligation assay and co-immunoprecipitation results confirmed interaction between ITGB4 and PXN in H2009 cells. By virtually screening a library of 1440 FDA-approved compounds that bind to the N-terminal region of PXN, the top 40 hits were screened using a cell-based assay with H358 cells. These results determined the top 11 compounds with the lowest IC50 values (0.8 nM to 1.54 μM). Drug sensitivity to these 11 compounds was tested again using the cisplatin-resistant cell line H2009. Carfilzomib, a proteasome inhibitor, was identified to be the most effective (IC50 = 193.4 nM). This compound was further tested in 3D cell line-derived spheroids and patient cancer tissue-derived organoids that were allowed to form in multi-well ultra-low attachment plates. These cultures were monitored in real-time using the IncuCyte S3 Live-Cell Analysis System. In cisplatin-resistant cell lines H2009 and H1993, carfilzomib was the most effective (IC50 = 193.4 nM, 1.77 μM, respectively), irrespective of KRAS status. Wound healing was impeded by 20% in H2009 with a submicromolar dose (200 nM). In both H2009 and H1993 cells, a sublethal dose of carfilzomib decreased expression of ITGB4 and phospho-Rb (S807/811) and increased expression of p27, γH2AX, and cleaved PARP, indicating cell cycle inhibition and cell death. In H2009 spheroids, carfilzomib (600 nM) was highly effective in decreasing spheroid viability by 60% and inducing a 2.5-fold increase in caspase-3/7-mediated apoptosis. Also in patient-derived organoids, carfilzomib induced an increase in apoptosis by 5-fold in one case and 50-fold in a second case compared to cisplatin. Another proteasome inhibitor ixazomib was among the top 11 compounds tested in H2009 cells but had a higher IC50 (2.93 μM) compared to carfilzomib, indicating greater efficacy of carfilzomib against cisplatin-resistant LUAD. These results impart a potential therapeutic approach for overcoming chemoresistance in LUAD by targeting the focal adhesion complex through repurposing carfilzomib, an FDA-approved compound. Citation Format: Arin Nam, Atish Mohanty, Supriyo Bhattacharya, Saumya Srivastava, Rajendra Pangeni, Dan Raz, John Orban, Prakash Kulkarni, Ravi Salgia. Carfilzomib: A potential drug against cisplatin-resistant lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6329.

Abstract 2607: Integrin Beta4/Paxillin mediated chemoresistance in lung adenocarcinoma

Abstract Introduction: Non-small cell lung cancer (NSCLC) accounts for 85% of the diagnosed lung cancers, of which 40% are lung adenocarcinoma (LUAD). These adenocarcinomas are often diagnosed with advanced or metastatic disease and are typically treated with a combination of cisplatin and immunotherapy. In most cases, malignant cells exposed to cisplatin adapt and become less susceptible to the anti-proliferative and cytotoxic effects of the drugs. Thus, many cisplatin-treated patients experience therapeutic failure and tumor recurrence. Therefore, there is a dire need for a deeper understanding of chemoresistance and the identification of prognostic and predictive markers to discern responders from non-responders. Here, we interrogate the role of Integrinβ4 (ITGB4) and Paxillin (PXN) in developing cisplatin resistance that are components of the focal adhesion complex. Experimental Procedures: Validated lung adenocarcinoma cancer cell lines were engineered to express nuclear specific red fluorescence protein for live tracking of cell proliferation, wound healing and cell death using InCucyte Live Cell Imaging System. Cell line-derived spheroids were generated to recapitulate in vivo conditions and spheroid integrity was monitored over time. Changes in gene expression were checked using western blotting or qPCR. Mitochondrial activity was analyzed using Seahorse and genomic stability was measured using γH2AX staining. Immunohistochemical analysis of the stained tissue section was done using the QuPath software. Summary: Cells with higher expression of PXN and ITGB4 are ~50% less sensitive to cisplatin as compared to low expressing cells. Silencing both PXN and ITGB4 in resistant cells inhibited their proliferation by 6-fold, migration by 30 - 40% and increase cisplatin sensitivity by ~ 60%, in 2D and 3D cultures. Differential gene expression analysis revealed significant enrichment of down regulated genes belonging to pathways like TargetsV1, G2-M checkpoint and E2F targets. VDAC1 and USP1 of MYC target V1 pathway were downregulated ~70% and ~ 50% respectively. Knocking down USP1 increased number of γH2AX foci counts, suggesting increased susceptibility to DNA damage. Knocking down VDAC1 increase mitochondrial respiration ~ 30% and increase ROS production by 3-fold. H3K27 acetylation dropped by ~70% at USP1 promoter site in double knocked down cells, inhibiting its transcription. Cisplatin treatment on a heterogeneous population enriched a fraction of cells expressing higher ITGB4 by 2-fold. Immunohistochemical analysis of tumor tissue sections revealed heterogeneity in expression of PXN, ITGB4 or both. Similar, data was also observed in exosomes isolated from patient serum. Conclusion: ITGB4 and PXN co-expression helps cancer cells to maintain their genomic stability and mitochondrial function, thus helping it to evade drug toxicity. NSCLC adenocarcinomas harboring high expression of them are likely to develop drug resistance. Citation Format: Atish Mohanty, Arin Nam, Alex Pozhitkov, Lu Yang, Michael Nelson, Xiwei Wu, Prakash Kulkarni, Erminia Massarelli, Ravi Salgia. Integrin Beta4/Paxillin mediated chemoresistance in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2607.