Teriflunomide/leflunomide synergize with chemotherapeutics by decreasing mitochondrial fragmentation via DRP1 in SCLC.

Although up to 80% small cell lung cancer (SCLC) patients' response is good for first-line chemotherapy regimen, most patients develop recurrence of the disease within weeks to months. Here, we report cytostatic effect of leflunomide (Leflu) and teriflunomide (Teri) on SCLC cell proliferation through inhibition of DRP1 phosphorylation at Ser616 and decreased mitochondrial fragmentation. When administered together, Teri and carboplatin (Carbo) act synergistically to significantly inhibit cell proliferation and DRP1 phosphorylation, reduce abundance of intermediates in pyrimidine de novo pathway, and increase apoptosis and DNA damage. Combination of Leflu&Carbo has anti-tumorigenic effect in vivo. Additionally, lurbinectedin (Lur) and Teri potently and synergistically inhibited spheroid growth and depleted uridine and DRP1 phosphorylation in mouse tumors. Our results suggest combinations of Carbo and Lur with Teri or Leflu are efficacious and underscore how the relationship between DRP1/DHODH and mitochondrial plasticity serves as a potential therapeutic target to validate these treatment strategies in SCLC clinical trials.

A Systems Biology Approach for Addressing Cisplatin Resistance in Non-Small Cell Lung Cancer.

Translational research in medicine, defined as the transfer of knowledge and discovery from the basic sciences to the clinic, is typically achieved through interactions between members across scientific disciplines to overcome the traditional silos within the community. Thus, translational medicine underscores 'Team Medicine', the partnership between basic science researchers and clinicians focused on addressing a specific goal in medicine. Here, we highlight this concept from a City of Hope perspective. Using cisplatin resistance in non-small cell lung cancer (NSCLC) as a paradigm, we describe how basic research scientists, clinical research scientists, and medical oncologists, in true 'Team Science' spirit, addressed cisplatin resistance in NSCLC and identified a previously approved compound that is able to alleviate cisplatin resistance in NSCLC. Furthermore, we discuss how a 'Team Medicine' approach can help to elucidate the mechanisms of innate and acquired resistance in NSCLC and develop alternative strategies to overcome drug resistance.

Addressing Drug Resistance in Cancer: A Team Medicine Approach.

Drug resistance remains one of the major impediments to treating cancer. Although many patients respond well initially, resistance to therapy typically ensues. Several confounding factors appear to contribute to this challenge. Here, we first discuss some of the challenges associated with drug resistance. We then discuss how a 'Team Medicine' approach, involving an interdisciplinary team of basic scientists working together with clinicians, has uncovered new therapeutic strategies. These strategies, referred to as intermittent or 'adaptive' therapy, which are based on eco-evolutionary principles, have met with remarkable success in potentially precluding or delaying the emergence of drug resistance in several cancers. Incorporating such treatment strategies into clinical protocols could potentially enhance the precision of delivering personalized medicine to patients. Furthermore, reaching out to patients in the network of hospitals affiliated with leading academic centers could help them benefit from such innovative treatment options. Finally, lowering the dose of the drug and its frequency (because of intermittent rather than continuous therapy) can also have a significant impact on lowering the toxicity and undesirable side effects of the drugs while lowering the financial burden carried by the patient and insurance providers.

Protein Phosphatase 2A as a Therapeutic Target in Small Cell Lung Cancer.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase involved in the regulation of apoptosis, proliferation, and DNA-damage response, is overexpressed in many cancers, including small cell lung cancer (SCLC). Here we report that LB100, a small molecule inhibitor of PP2A, when combined with platinum-based chemotherapy, synergistically elicited an antitumor response both in vitro and in vivo with no apparent toxicity. Using inductively coupled plasma mass spectrometry, we determined quantitatively that sensitization via LB100 was mediated by increased uptake of carboplatin in SCLC cells. Treatment with LB100 alone or in combination resulted in inhibition of cell viability in two-dimensional culture and three-dimensional spheroid models of SCLC, reduced glucose uptake, and attenuated mitochondrial and glycolytic ATP production. Combining LB100 with atezolizumab increased the capacity of T cells to infiltrate and kill tumor spheroids, and combining LB100 with carboplatin caused hyperphosphorylation of the DNA repair marker γH2AX and enhanced apoptosis while attenuating MET signaling and invasion through an endothelial cell monolayer. Taken together, these data highlight the translational potential of inhibiting PP2A with LB100 in combination with platinum-based chemotherapy and immunotherapy in SCLC.

Inhibiting crosstalk between MET signaling and mitochondrial dynamics and morphology: a novel therapeutic approach for lung cancer and mesothelioma.

The receptor tyrosine kinase MET is frequently involved in malignant transformation and inhibiting its activity in MET-dependent cancers is associated with improved clinical outcomes. Emerging evidence also suggests that mitochondria play an essential role in tumorigenesis and Dynamin Related Protein (DRP1), a key component of the mitochondrial fission machinery, has emerged as an attractive therapeutic target. Here, we report that inhibiting MET activity with the tyrosine kinase inhibitor MGCD516 attenuates viability, migration, and invasion of non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines in vitro, and significantly retards tumor growth in vivo. Interestingly, MGCD516 treatment also results in altered mitochondrial morphology in these cell lines. Furthermore, inhibiting MET pharmacologically or knocking down its expression using siRNA, decreases DRP1 activity alluding to possible crosstalk between them in these two cancers. Consistently, a combination of MGCD516 and mdivi-1, a quinazolinone reported to inhibit mitochondrial fission, is more effective in attenuating proliferation of NSCLC and MPM cell lines than either drug alone. Considered together, the present study has uncovered a novel mechanism underlying mitochondrial regulation by MET that involves crosstalk with DRP1, and suggests that a combination therapy targeting both MET and DRP1 could be a novel strategy for NSCLC and MPM.

Exosomal miRNAs species in the blood of small cell and non-small cell lung cancer patients.

Lung cancer is a devastating disease with overall bleak prognosis. Current methods to diagnose lung cancer are rather invasive and are inadequate to detect the disease at an early stage when treatment is likely to be most effective. In this study, a shotgun sequencing approach was used to study the microRNA (miRNA) cargo of serum-derived exosomes of small cell lung cancer (SCLC) (n=9) and non-small cell lung cancer (NSCLC) (n=11) patients, and healthy controls (n=10). The study has identified 17 miRNA species that are differentially expressed in cancer patients and control subjects. Furthermore, within the patient groups, a set of miRNAs were differentially expressed in exosomal samples obtained before and after chemotherapy treatment. This manuscript demonstrates the potential of exosomal miRNAs for developing noninvasive tests for disease differentiation and treatment monitoring in lung cancer patients.

Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma.

The non-receptor cytoplasmic tyrosine kinase, Focal Adhesion Kinase (FAK) is known to play a key role in a variety of normal and cancer cellular functions such as survival, proliferation, migration and invasion. It is highly active and overexpressed in various cancers including Pancreatic Ductal Adenocarcinoma (PDAC) and Malignant Pleural Mesothelioma (MPM). Here, initially, we demonstrate that FAK is overexpressed in both PDAC and MPM cell lines. Then we analyze effects of two small molecule inhibitors PF-573228, and PF-431396, which are dual specificity inhibitors of FAK and proline rich tyrosine kinase 2 (PYK2), as well as VS-6063, another small molecule inhibitor that specifically inhibits FAK but not PYK2 for cell growth, motility and invasion of PDAC and MPM cell lines. Treatment with PF-573228, PF-431396 and VS-6063 cells resulted in a dose-dependent inhibition of growth and anchorage-independent colony formation in both cancer cell lines. Furthermore, these compounds suppressed the phosphorylation of FAK at its active site, Y397, and functionally induced significant apoptosis and cell cycle arrest in both cell lines. Using the ECIS (Electric cell-substrate impedance sensing) system, we found that treatment of both PF compounds suppressed adherence and migration of PDAC cells on fibronectin. Interestingly, 3D-tumor organoids derived from autochthonous KC (Kras;PdxCre) mice treated with PF-573228 revealed a significant decrease in tumor organoid size and increase in organoid cell death. Taken together, our results show that FAK is an important target for mesothelioma and pancreatic cancer therapy that merit further translational studies.

PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent.

Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity.

Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6-24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity.

HABP2 is a Novel Regulator of Hyaluronan-Mediated Human Lung Cancer Progression.

BACKGROUND: Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA), which we have previously demonstrated can regulate the activity of the extracellular serine protease, hyaluronan binding protein 2 (HABP2). This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics. METHODS: Immunohistochemical analysis was performed on lung cancer patient samples using anti-HABP2 antibody. Stable control, shRNA, and HABP2 overexpressing human lung adenocarcinoma cells were evaluated using immunoblot analysis, migration, extravasation, and urokinase plasminogen activator (uPA) activation assays with or without high-molecular weight HA or low-molecular weight HA (LMW-HA). In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. RESULTS: We provide evidence that HABP2 is an important regulator of lung cancer progression. HABP2 expression was increased in several subtypes of patient non-small cell lung cancer samples. Further, HABP2 overexpression increased LMW-HA-induced uPA activation, migration, and extravasation in human lung adenocarcinoma cells. In vivo, overexpression of HABP2 in human lung adenocarcinoma cells increased primary tumor growth rates in nude mice by ~2-fold and lung metastasis by ~10-fold compared to vector control cells (n = 5/condition). CONCLUSION: Our data suggest a possible direct effect of HABP2 on uPA activation and lung cancer progression. Our observations suggest that exploration of HABP2 in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

Transactivation of the receptor-tyrosine kinase ephrin receptor A2 is required for the low molecular weight hyaluronan-mediated angiogenesis that is implicated in tumor progression.

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.

GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids.

Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell-cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane-localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane-localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.

The Mu opioid receptor promotes opioid and growth factor-induced proliferation, migration and Epithelial Mesenchymal Transition (EMT) in human lung cancer.

Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR) can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC) cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT) in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation) which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA) or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05). Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.

Overexpression of the µ-opioid receptor in human non-small cell lung cancer promotes Akt and mTOR activation, tumor growth, and metastasis.

BACKGROUND: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that µ-opioid agonists can influence cancer progression. Based on our previous studies indicating the µ-opioid receptor (MOR) is up-regulated in several types of non-small cell lung cancer, this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. METHODS: Stable vector control and MOR1 overexpressing human bronchioloalveolar carcinoma cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus), or the peripheral MOR antagonist, methylnaltrexone. In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in lungs of the nude mouse model. RESULTS: The authors provide evidence that MOR is an important regulator of lung cancer progression. MOR overexpression increased Akt and mTOR activation, proliferation, and extravasation in human bronchioloalveolar carcinoma cells. In vivo, overexpression of MOR in human bronchoalveolar carcinoma cells increased primary tumor growth rates in nude mice by approximately 2.5-fold and lung metastasis by approximately 20-fold compared with vector control cells (n = 4 per condition). CONCLUSIONS: The overexpression data suggest a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. The authors' observations further suggest that exploration of MOR in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

Hyaluronic Acid binding protein 2 is a novel regulator of vascular integrity.

OBJECTIVE: The disruption of the endothelial cell barrier is a critical feature of inflammation and an important contributing factor to acute lung injury (ALI), an inflammatory condition that is a major cause of morbidity and mortality in critically ill patients. We evaluated the role of the extracellular serine protease, hyaluronic acid binding protein 2 (HABP2), in vascular barrier regulation. METHODS AND RESULTS: By using immunoblot and immunohistochemical analysis, we observed that lipopolysaccharide (LPS) induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells (ECs) in vitro. High-molecular-weight hyaluronan (HMW-HA, approximately 1x10(6) Da) decreased HABP2 protein expression in human pulmonary microvascular ECs and decreased purified HABP2 enzymatic activity, whereas low-molecular-weight HA (LMW-HA, approximately 2500 Da) increased these activities. The effects of LMW-HA, but not HMW-HA, on HABP2 activity were inhibited with a peptide of the polyanion-binding domain of HABP2. Silencing (small interfering RNA) HABP2 expression augmented HMW-HA-induced EC barrier enhancement and inhibited LPS and LMW-HA-mediated EC barrier disruption, results that were reversed with overexpression of HABP2. Silencing protease-activated receptor 1 and 3, RhoA, or Rho kinase expression attenuated LPS-, LMW-HA-, and HABP2-mediated EC barrier disruption. By using murine models of acute lung injury, we observed that LPS- and ventilator-induced pulmonary vascular hyperpermeability was significantly reduced with vascular silencing (small interfering RNA) of HABP2. CONCLUSIONS: HABP2 negatively regulates vascular integrity via activation of protease-activated receptor/RhoA/Rho kinase signaling and represents a potentially useful therapeutic target for syndromes of increased vascular permeability.

Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction.

2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.