A new long noncoding RNA (lncRNA) is induced in cutaneous squamous cell carcinoma and down-regulates several anticancer and cell differentiation genes in mouse.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Although some of the early events involved in this pathology have been identified, the subsequent steps leading to tumor development are poorly defined. We demonstrate here that the development of mouse tumors induced by the concomitant application of a carcinogen and a tumor promoter (7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively) is associated with the up-regulation of a previously uncharacterized long noncoding RNA (lncRNA), termed AK144841. We found that AK144841 expression was absent from normal skin and was specifically stimulated in tumors and highly tumorigenic cells. We also found that AK144841 exists in two variants, one consisting of a large 2-kb transcript composed of four exons and one consisting of a 1.8-kb transcript lacking the second exon. Gain- and loss-of-function studies indicated that AK144841 mainly inhibited gene expression, specifically down-regulating the expression of genes of the late cornified envelope-1 (Lce1) family involved in epidermal terminal differentiation and of anticancer genes such as Cgref1, Brsk1, Basp1, Dusp5, Btg2, Anpep, Dhrs9, Stfa2, Tpm1, SerpinB2, Cpa4, Crct1, Cryab, Il24, Csf2, and Rgs16 Interestingly, the lack of the second exon significantly decreased AK144841's inhibitory effect on gene expression. We also noted that high AK144841 expression correlated with a low expression of the aforementioned genes and with the tumorigenic potential of cell lines. These findings suggest that AK144841 could contribute to the dedifferentiation program of tumor-forming keratinocytes and to molecular cascades leading to tumor development.

Neutrophil extracellular traps formed during chemotherapy confer treatment resistance via TGF-ß activation.

Metastasis is the major cause of cancer death, and the development of therapy resistance is common. The tumor microenvironment can confer chemotherapy resistance (chemoresistance), but little is known about how specific host cells influence therapy outcome. We show that chemotherapy induces neutrophil recruitment and neutrophil extracellular trap (NET) formation, which reduces therapy response in mouse models of breast cancer lung metastasis. We reveal that chemotherapy-treated cancer cells secrete IL-1ß, which in turn triggers NET formation. Two NET-associated proteins are required to induce chemoresistance: integrin-αvß1, which traps latent TGF-ß, and matrix metalloproteinase 9, which cleaves and activates the trapped latent TGF-ß. TGF-ß activation causes cancer cells to undergo epithelial-to-mesenchymal transition and correlates with chemoresistance. Our work demonstrates that NETs regulate the activities of neighboring cells by trapping and activating cytokines and suggests that chemoresistance in the metastatic setting can be reduced or prevented by targeting the IL-1ß-NET-TGF-ß axis.

Targeting of c-MET and AXL by cabozantinib is a potential therapeutic strategy for patients with head and neck cell carcinoma.

Local or metastatic relapse following surgery, radiotherapy, and cisplatin is the leading cause of death in patients with head and neck squamous cell carcinoma (HNSCC). Our study shows overexpression of c-MET and AXL in HNSCC cells and patients resistant to radiotherapy and cisplatin. We demonstrate that cabozantinib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), c-MET, and AXL, decreases migration, invasion, and proliferation and induces mitotic catastrophe and apoptotic cell death of naive and radiotherapy- and cisplatin-resistant HNSCC cells. Cabozantinib inhibits the growth and metastatic spread of experimental HNSCC in zebrafish and the growth of experimental HNSCC in mice by blocking tumor cell proliferation and angiogenesis. The efficacy of cabozantinib is also confirmed on viable sections of surgically removed specimens of human HNSCC and on a patient who relapses after five lines of treatment. These results suggest that cabozantinib is relevant for the treatment of patients with HNSCC after relapse under radiotherapy and cisplatin.

The Polo-like kinase 1 inhibitor onvansertib represents a relevant treatment for head and neck squamous cell carcinoma resistant to cisplatin and radiotherapy.

Rationale: Head and neck squamous cell carcinoma (HNSCC) represent the 4th most aggressive cancer. 50% of patients relapse to the current treatments combining surgery, radiotherapy and cisplatin and die two years after the diagnosis. Elevated expression of the polo-like kinase 1 (Plk1) correlated to a poor prognosis in epidermoid carcinomas. Methods: The molecular links between Plk1 and resistance to cisplatin/radiotherapy were investigated in patients and cell lines resistant to cisplatin and/or to radiotherapy. The therapeutic relevance of the Plk1 inhibitor onvansertib, alone or combined with cisplatin/radiotherapy, was evaluated on the proliferation/migration on HNSCC cell lines, in experimental HNSCC in mice, in a zebrafish metastasis model and on patient-derived 3D tumor sections. Results: Plk1 expression correlated to a bad prognosis in HNSCC and increased after relapse on cisplatin/radiotherapy. Onvansertib induced mitotic arrest, chromosomic abnormalities and polyploidy leading to apoptosis of sensitive and resistant HNSCC cells at nanomolar concentrations without any effects on normal cells. Onvansertib inhibited the growth of experimental HNSCC in mice and metastatic dissemination in zebrafishes. Moreover, onvansertib combined to cisplatin and/or radiotherapy resulted in a synergic induction of tumor cell death. The efficacy of onvansertib alone and in combination with reference treatments was confirmed on 3D viable sections of HNSCC surgical specimens. Conclusions: Targeting Plk1 by onvansertib represents a new strategy for HNSCC patients at the diagnosis in combination with reference treatments, or alone as a second line treatment for HNCSCC patients experiencing relapses.

Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy.

Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.

Matrix Stiffening and EGFR Cooperate to Promote the Collective Invasion of Cancer Cells.

In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg Cancer Res; 78(18); 5229-42. ©2018 AACR.

Membrane-bound ICAM-1 contributes to the onset of proinvasive tumor stroma by controlling acto-myosin contractility in carcinoma-associated fibroblasts.

Acto-myosin contractility in carcinoma-associated fibroblasts leads to assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock-down of LIF-dependent genes in fibroblasts, we identified the glycoprotein ICAM-1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that the membrane-bound ICAM-1 isoform is necessary and sufficient to promote inflammation-dependent extracellular matrix contraction, which favors cancer cell invasion. Indeed, ICAM-1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM-1 expression by establishing a positive feedback signaling. Thus, targeting stromal ICAM-1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination.

Lack of CT scanner in a rural emergency department increases inter-facility transfers: a pilot study.

OBJECTIVE: Rural emergency departments (EDs) are an important gateway to care for the 20% of Canadians who reside in rural areas. Less than 15% of Canadian rural EDs have access to a computed tomography (CT) scanner. We hypothesized that a significant proportion of inter-facility transfers from rural hospitals without CT scanners are for CT imaging. Our objective was to assess inter-facility transfers for CT imaging in a rural ED without a CT scanner. RESULTS: We selected a rural ED that offers 24/7 medical care with admission beds but no CT scanner. Descriptive statistics were collected from 2010 to 2015 on total ED visits and inter-facility transfers. Data was accessible through hospital and government databases. Between 2010 and 2014, there were respectively 13,531, 13,524, 13,827, 12,883, and 12,942 ED visits, with an average of 444 inter-facility transfers. An average of 33% (148/444) of inter-facility transfers were to a rural referral centre with a CT scan, with 84% being for CT scan. Inter-facility transfers incur costs and potential delays in patient diagnosis and management, yet current databases could not capture transfer times. Acquiring a CT scan may represent a reasonable opportunity for the selected rural hospital considering the number of required transfers.

Down-regulation of Hox A7 is required for cell adhesion and migration on fibronectin during early HL-60 monocytic differentiation.

Hox genes, which are key regulators of cell fate and pattern formation during embryogenesis, are also important regulators of hematopoiesis, and different combinations of Hox gene products are involved in lineage commitment or maturation. However, their molecular and cellular modes of action are not yet completely understood. Recent studies have indicated that Hox genes are involved in the regulation of cell-extracellular matrix (ECM) interactions and cell migration. Here, we report that Hox A7, a gene frequently overexpressed in acute myeloid leukemia, is down-regulated during HL-60 monocytic differentiation. Using a model in which HL-60 cells are induced to differentiate toward the monocytic lineage with bone marrow stromal-like cells, we demonstrate that Hox A7-sustained expression disturbs the regulation of cell adhesive and migratory capacities on fibronectin during early differentiation. We show that this is accompanied by a partial blockage of the transcriptional induction of proline-rich tyrosine kinase 2, a gene coding for a focal adhesion kinase active in monocytes, and of tissue transglutaminase, a gene coding for a fibronectin coreceptor in monocytes. This is the first report that demonstrates the involvement of a Hox gene in the regulation of adhesion and migration of hematopoietic cells and that links it to the deregulation of genes involved in cell-ECM interactions and downstream signaling pathways.

Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts.

Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.

Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation.

BACKGROUND: Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells. RESULTS: Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 (-/-) MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 (-/-) MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 (-/-) MEF tumors grew faster than wild-type MEF tumors. CONCLUSIONS: Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.

Cross-talk between RhoGTPases and stress activated kinases for matrix metalloproteinase-9 induction in response to keratinocytes injury.

Cell migration and extracellular matrix remodeling are two essential processes of wound healing, regulated by extracellular metalloproteinases such as matrix metalloproteinase-2 (Gelatinase A) and matrix metalloproteinase-9 (Gelatinase B). Expression of matrix metalloproteinase-9 is deregulated in numerous wound healing pathologies. To date the mechanisms regulating matrix metalloproteinase-9 during normal wound healing are poorly documented. Using both primary cultures of normal human keratinocytes and a wounding device especially designed to dissect the molecular events during the healing process in vitro, we show that matrix metalloproteinase-9 is stimulated by injury in normal human keratinocytes. This upregulation results from the mechanical stress created by injury and not from a soluble factor, secreted by wounded normal human keratinocytes. We also demonstrate that the Rho family of small GTPases, p38[MAPK] and JNK together play a key part in the signaling pathways controlling the stimulation of matrix metalloproteinase-9 in wounded cells. We provide lines of evidence indicating that in wounded keratinocytes, upregulation of matrix metalloproteinase-9 depends on two distinct pathways. The first involves Rac1 and/or Cdc42 that control the activation of p38[MAPK]. The second depends on RhoA activation that is required for stimulation of JNK.

LIF mediates proinvasive activation of stromal fibroblasts in cancer.

Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF) as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA) expression. We demonstrate that a pulse of transforming growth factor ß (TGF-ß) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-ß-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor) counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

HIF1 transcription factor regulates laminin-332 expression and keratinocyte migration.

Epidermal wound repair is a complex process involving the fine orchestrated regulation of crucial cell functions, such as proliferation, adhesion and migration. Using an in vitro model that recapitulates central aspects of epidermal wound healing, we demonstrate that the transcription factor HIF1 is strongly stimulated in keratinocyte cultures submitted to mechanical injury. Signals generated by scratch wounding stabilise the HIF1alpha protein, which requires activation of the PI3K pathway independently of oxygen availability. We further show that upregulation of HIF1alpha plays an essential role in keratinocyte migration during the in vitro healing process, because HIF1alpha inhibition dramatically delays the wound closure. In this context, we demonstrate that HIF1 controls the expression of laminin-332, one of the major epithelial cell adhesion ligands involved in cell migration and invasion. Indeed, silencing of HIF1alpha abrogates injury-induced laminin-332 expression, and we provide evidence that HIF1 directly regulates the promoter activity of the laminin alpha3 chain. Our results suggest that HIF1 contributes to keratinocyte migration and thus to the re-epithelialisation process by regulating laminin-332.

hCOX18 and hCOX19: two human genes involved in cytochrome c oxidase assembly.

We identified the human homologues of yCOX18 and yCOX19, two Saccharomyces cerevisiae genes involved in the biogenesis of mitochondrial respiratory chain complexes. In yeast, these two genes are required for the expression of cytochrome c oxidase: Cox18p catalyses the insertion of Cox2p COOH-tail into the mitochondrial inner membrane, and Cox19p is probably involved in metal transport to the intermembrane space. Both hCox18p and hCox19p present significant amino acid identity with the corresponding yeast polypeptides and reveal highly conserved functional domains. In addition, their subcellular localization is analogous to that of the yeast proteins. These data strongly suggest that the human gene products share similar functions with their yeast homologues. These two COX-assembly genes represent new candidates for mutational analysis in patients with isolated COX deficiency of unknown etiology.

Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration.

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.