Defining melanoma combination therapies that provide senolytic sensitivity in human melanoma cells.

Malignant Melanoma that resists immunotherapy remains the deadliest form of skin cancer owing to poor clinically lasting responses. Alternative like genotoxic or targeted chemotherapy trigger various cancer cell fates after treatment including cell death and senescence. Senescent cells can be eliminated using senolytic drugs and we hypothesize that the targeted elimination of therapy-induced senescent melanoma cells could complement both conventional and immunotherapies. We utilized a panel of cells representing diverse mutational background relevant to melanoma and found that they developed distinct senescent phenotypes in response to treatment. A genotoxic combination therapy of carboplatin-paclitaxel or irradiation triggered a mixed response of cell death and senescence, irrespective of BRAF mutation profiles. DNA damage-induced senescent melanoma cells exhibited morphological changes, residual DNA damage, and increased senescence-associated secretory phenotype (SASP). In contrast, dual targeted inhibition of Braf and Mek triggered a different mixed cell fate response including senescent-like and persister cells. While persister cells could reproliferate, senescent-like cells were stably arrested, but without detectable DNA damage and senescence-associated secretory phenotype. To assess the sensitivity to senolytics we employed a novel real-time imaging-based death assay and observed that Bcl2/Bcl-XL inhibitors and piperlongumine were effective in promoting death of carboplatin-paclitaxel and irradiation-induced senescent melanoma cells, while the mixed persister cells and senescent-like cells resulting from Braf-Mek inhibition remained unresponsive. Interestingly, a direct synergy between Bcl2/Bcl-XL inhibitors and Braf-Mek inhibitors was observed when used out of the context of senescence. Overall, we highlight diverse hallmarks of melanoma senescent states and provide evidence of context-dependent senotherapeutics that could reduce treatment resistance while also discussing the limitations of this strategy in human melanoma cells.

Premature thymic functional senescence is a hallmark of childhood acute lymphoblastic leukemia survivorship.

Childhood acute lymphoblastic leukemia (cALL) survivors suffer early-onset chronic diseases classically associated with aging. Normal aging is accompanied by organ dysfunctions, including immunological ones. We hypothesize that thymic immunosenescence occurs in cALL survivors and that its severity may correlate with early-onset chronic diseases. The PETALE study is a cALL survivor cohort with an extensive cardiovascular and metabolic evaluation. The thymic immunosenescence biomarker, signal joint T-cell receptor excision circles (TREC), was evaluated and was highly correlated with age in healthy participants (n = 281) and cALL survivors (n = 248). We observed a systematic thymic immunoage accentuation in each cALL survivor compared to controls ranging from 5.9 to 88.3 years. The immunoage gain was independent of age at diagnosis and treatment modalities and was more severe for females. Thymic aging was associated with several pathophysiological parameters, was greater in survivors suffering from metabolic syndrome, but there was no significant association with global physical condition. The decrease in TREC was independent from blood cell counts, which were normal, suggesting a segmental aging of the thymic compartment. Indeed, increased plasmatic T cell regulatory cytokines IL-6, IL-7 and GM-CSF accompanied high immunoage gain. Our data reveal that cALL or its treatment trigger a rapid immunoage gain followed by further gradual thymic immunosenescence, similar to normal aging. This leads to an enduring shift in accentuated immunoage compared to chronological age. Thus, accentuated thymic immunosenescence is a hallmark of cALL survivorship and TREC levels could be useful immunosenescence biomarkers to help monitoring the health of cancer survivors.

Dynamic and scalable assessment of the senescence-associated secretory phenotype (SASP).

Dual-faced cellular senescence is responsible for beneficial biological processes and for age-related pathologies. Senescent cells under stable proliferation arrest develop numerous senescence-associated phenotypes such as the potent pro-inflammatory secretome called the senescence-associated secretory phenotype (SASP). The SASP shapes the senescent microenvironment and influences the biology of adjacent cells, including the modulation of proliferation and migration/invasion, reinforcement/induction of peripheral senescence, and immune cell activity or recruitment. The SASP is a dynamic process with multiple waves of secreted factors described to interlace over a period of many days. Whether the senescence phenotype reaches a mature stable state remains controversial. Overall, the complexity of the context-dependent and timely SASP compositions and its varied microenvironmental impact demonstrate the importance of properly assessing SASP over time. In this chapter, we focus on scalable and dynamic experimental procedures to prepare SASP conditioned medium over time from cells receiving senescence-inducing stimuli. This SASP-containing conditioned medium can be used to assess the composition of the SASP, study SASP-related signaling pathways or evaluate the paracrine microenvironmental impact of senescent cells.

Radiotherapy on-chip: microfluidics for translational radiation oncology.

The clinical importance of radiotherapy in the treatment of cancer patients justifies the development and use of research tools at the fundamental, pre-clinical, and ultimately clinical levels, to investigate their toxicities and synergies with systemic agents on relevant biological samples. Although microfluidics has prompted a paradigm shift in drug discovery in the past two decades, it appears to have yet to translate to radiotherapy research. However, the materials, dimensions, design versatility and multiplexing capabilities of microfluidic devices make them well-suited to a variety of studies involving radiation physics, radiobiology and radiotherapy. This review will present the state-of-the-art applications of microfluidics in these fields and specifically highlight the perspectives offered by radiotherapy on-a-chip in the field of translational radiobiology and precision medicine. This body of knowledge can serve both the microfluidics and radiotherapy communities by identifying potential collaboration avenues to improve patient care.

Targeted Anti-Cancer Provascular Therapy Using Ultrasound, Microbubbles, and Nitrite to Increase Radiotherapy Efficacy.

Hypoxia is an important mechanism of resistance to radiation therapy in many human malignancies including prostate cancer. It has been recently shown that ultrasound targeted microbubble cavitation (UTMC) can increase blood perfusion in skeletal muscle by triggering nitric oxide signaling. Interestingly, this effect was amplified with a sodium nitrite coinjection. Since sodium nitrite has been shown to synergize with radiotherapy (RT), we hypothesized that UTMC with a sodium nitrite coinjection could further radiosensitize solid tumors by increasing blood perfusion and thus reduce tumor hypoxia. We evaluated (1) the ability of UTMC with and without nitrite to increase perfusion in muscle (mouse hindlimbs) and human prostate tumors using different pulse lengths and pressure; (2) the efficacy of this approach as a provascular therapy given directly before RT in the human prostate subcutaneous xenografts PC3 tumor model. Using long pulses with various pressures, in muscle, the provascular response following UTMC was strong (6.61 ± 4.41-fold increase in perfusion post-treatment). In tumors, long pulses caused an increase in perfusion (2.42 ± 1.38-fold) at lower mechanical index (MI = 0.25) but not at higher MI (0.375, 0.5, and 0.750) when compared to control (no UTMC). However, when combined with RT, UTMC with long pulses (MI = 0.25) did not improve tumor growth inhibition. With short pulses, in muscle, the provascular response following UTMC (SONOS) + nitrite was strong (13.74 ± 8.60-fold increase in perfusion post-treatment). In tumors, UTMC (SONOS) + nitrite also caused a provascular response (1.94 ± 1.20-fold increase in perfusion post-treatment) that lasted for at least 10 min, but not with nitrite alone. Interestingly, the blunted provascular response observed for long pulses at higher MI without nitrite was reversed with the addition of nitrite. UTMC (SONOS) with and without nitrite caused an increase in perfusion in tumors. The provascular response observed for UTMC (SONOS) + nitrite was confirmed by histology. Finally, there was an improved growth inhibition for the 8 Gy RT dose + nitrite + UTMC group vs 8 Gy RT + nitrite alone. This effect was not significant with mice treated by UTMC + nitrite and receiving doses of 0 or 2 Gy RT. In conclusion, UTMC + nitrite increased blood flow leading to an increased efficacy of higher doses of RT in our tumor model, warranting further study of this strategy.

Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-ß-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence.

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.

NCOR1 Sustains Colorectal Cancer Cell Growth and Protects against Cellular Senescence.

NCOR1 is a corepressor that mediates transcriptional repression through its association with nuclear receptors and specific transcription factors. Some evidence supports a role for NCOR1 in neonatal intestinal epithelium maturation and the maintenance of epithelial integrity during experimental colitis in mice. We hypothesized that NCOR1 could control colorectal cancer cell proliferation and tumorigenicity. Conditional intestinal epithelial deletion of Ncor1 in ApcMin/+ mice resulted in a significant reduction in polyposis. RNAi targeting of NCOR1 in Caco-2/15 and HT-29 cell lines led to a reduction in cell growth, characterized by cellular senescence associated with a secretory phenotype. Tumor growth of HT-29 cells was reduced in the absence of NCOR1 in the mouse xenografts. RNA-seq transcriptome profiling of colon cancer cells confirmed the senescence phenotype in the absence of NCOR1 and predicted the occurrence of a pro-migration cellular signature in this context. SOX2, a transcription factor essential for pluripotency of embryonic stem cells, was induced under these conditions. In conclusion, depletion of NCOR1 reduced intestinal polyposis in mice and caused growth arrest, leading to senescence in human colorectal cell lines. The acquisition of a pro-metastasis signature in the absence of NCOR1 could indicate long-term potential adverse consequences of colon-cancer-induced senescence.

DCBLD1 is associated with the integrin signaling pathway and has prognostic value in non-small cell lung and invasive breast carcinoma.

Germline single nucleotide polymorphisms in the promoter region of the DCBLD1 gene are associated with non-smoking cases of both non-small cell lung carcinoma (NSCLC) and human papillomavirus-negative head and neck cancer. However the clinical relevance and function of DCBLD1 remain unclear. This multicenter retrospective study was designed to evaluate the prognostic value and function of DCBLD1 in the four main solid cancers: NSCLC, invasive breast carcinoma, colorectal adenocarcinoma and prostate adenocarcinoma. We included the following cohorts: GSE81089 NSCLC, METABRIC invasive breast carcinoma, GSE14333 colorectal adenocarcinoma, GSE70770 prostate adenocarcinoma and The Cancer Genome Atlas (TCGA) Firehose Legacy cohorts of all four cancers. DCBLD1 gene expression was associated with a worse overall survival in multivariate analyses for both NSCLC cohorts (TCGA: P = 0.03 and GSE81089: P = 0.04) and both invasive breast carcinoma cohorts (TCGA: P = 0.02 and METABRIC: P < 0.001). Patients with high DCBLD1 expression showed an upregulation of the integrin signaling pathway in comparison to those with low DCBLD1 expression in the TCGA NSCLC cohort (FDR = 5.16 × 10-14) and TCGA invasive breast carcinoma cohort (FDR = 1.94 × 10-05).

mTOR as a senescence manipulation target: A forked road.

Cellular senescence, cancer and aging are highly interconnected. Among many important molecular machines that lie at the intersection of this triad, the mechanistic (formerly mammalian) target of rapamycin (mTOR) is a central regulator of cell metabolism, proliferation, and survival. The mTOR signaling cascade is essential to maintain cellular homeostasis in normal biological processes or in response to stress, and its dysregulation is implicated in the progression of many disorders, including age-associated diseases. Accordingly, the pharmacological implications of mTOR inhibition using rapamycin or others rapalogs span the treatment of various human diseases from immune disorders to cancer. Importantly, rapamycin is one of the only known pan-species drugs that can extend lifespan. The molecular and cellular mechanisms explaining the phenotypic consequences of mTOR are vast and heavily studied. In this review, we will focus on the potential role of mTOR in the context of cellular senescence, a tumor suppressor mechanism and a pillar of aging. We will explore the link between senescence, autophagy and mTOR and discuss the opportunities to exploit senescence-associated mTOR functions to manipulate senescence phenotypes in age-associated diseases and cancer treatment.

Senolytic Targeting of Bcl-2 Anti-Apoptotic Family Increases Cell Death in Irradiated Sarcoma Cells.

Radiotherapy (RT) is a key component of cancer treatment. Most of the time, radiation is given after surgery but for soft-tissue sarcomas (STS), pre-surgical radiation is commonly utilized. However, despite improvements in RT accuracy, the rate of local recurrence remains high and is the major cause of death for patients with STS. A better understanding of cell fates in response to RT could provide new therapeutic options to enhance tumour cell killing by RT and facilitate surgical resection. Here, we showed that irradiated STS cell cultures do not die but instead undergo therapy-induced senescence (TIS), which is characterized by proliferation arrest, senescence-associated ß-galactosidase activity, secretion of inflammatory cytokines and persistent DNA damage. STS-TIS was also associated with increased levels of the anti-apoptotic Bcl-2 family of proteins which rendered cells targetable using senolytic Bcl-2 inhibitors. As oppose to radiation alone, the addition of senolytic agents Venetoclax (ABT-199) or Navitoclax (ABT-263) after irradiation induced a rapid apoptotic cell death in STS monolayer cultures and in a more complex three-dimensional culture model. Together, these data suggest a new promising therapeutic approach for sarcoma patients who receive neoadjuvant RT. The addition of senolytic agents to radiation treatments may significantly reduce tumour volume prior to surgery and thereby improve the clinical outcome of patients.

UM171-Expanded Cord Blood Transplants Support Robust T Cell Reconstitution with Low Rates of Severe Infections.

Rapid T cell reconstitution following hematopoietic stem cell transplantation (HSCT) is essential for protection against infections and has been associated with lower incidence of chronic graft-versus-host disease (cGVHD), relapse, and transplant-related mortality (TRM). While cord blood (CB) transplants are associated with lower rates of cGVHD and relapse, their low stem cell content results in slower immune reconstitution and higher risk of graft failure, severe infections, and TRM. Recently, results of a phase I/II trial revealed that single UM171-expanded CB transplant allowed the use of smaller CB units without compromising engraftment (www.clinicaltrials.gov, NCT02668315). We assessed T cell reconstitution in patients who underwent transplantation with UM171-expanded CB grafts and retrospectively compared it to that of patients receiving unmanipulated CB transplants. While median T cell dose infused was at least 2 to 3 times lower than that of unmanipulated CB, numbers and phenotype of T cells at 3, 6, and 12 months post-transplant were similar between the 2 cohorts. T cell receptor sequencing analyses revealed that UM171 patients had greater T cell diversity and higher numbers of clonotypes at 12 months post-transplant. This was associated with higher counts of naive T cells and recent thymic emigrants, suggesting active thymopoiesis and correlating with the demonstration that UM171 expands common lymphoid progenitors in vitro. UM171 patients also showed rapid virus-specific T cell reactivity and significantly reduced incidence of severe infections. These results suggest that UM171 patients benefit from rapid T cell reconstitution, which likely contributes to the absence of moderate/severe cGVHD, infection-related mortality, and late TRM observed in this cohort.

Biomarkers of cardiometabolic complications in survivors of childhood acute lymphoblastic leukemia.

Survivors of childhood acute lymphoblastic leukemia (cALL) are at higher risk of developing cardiometabolic complications. We aimed at exploring the associations between biomarkers of inflammation, oxidative stress, endothelial function, endotoxemia and cardiometabolic risk factors. We conducted a cross-sectional analysis in 246 cALL survivors (mean age, 22.1 ± 6.3 years; mean time since diagnosis, 15.5 ± 5.2 years) and evaluated the associations using a series of logistic regressions. Using structural equation models, we also tested if the relationship between endotoxemia and cardiometabolic complications was mediated by the latent (unobserved) variable inflammation inferred from the observed biomarkers CRP, TNF-α and IL-6. High leptin-adiponectin ratio was associated with obesity [adjusted OR = 15.7; 95% CI (6.2-39.7)], insulin resistance [20.6 (5.2-82.1)] and the metabolic syndrome [11.2 (2.6-48.7)]. Higher levels of plasminogen activator inhibitor-1 and tumor necrosis factor-α were associated with obesity [3.37 (1.6-7.1) and 2.34 (1.3-4.2), respectively] whereas high C-reactive protein levels were associated with insulin resistance [3.3 (1.6-6.8)], dyslipidemia [2.6 (1.4-4.9)] and MetS [6.5 (2.4-17.9)]. Our analyses provided evidence for a directional relationship between lipopolysaccharide binding protein, related to metabolic endotoxemia, inflammation and cardiometabolic outcomes. Identification of biomarkers and biological mechanisms could open new avenues for prevention strategies to minimize the long-term sequelae, improve follow-up and optimize the quality of life of this high-risk population.

Dual Inhibition of Autophagy and PI3K/AKT/MTOR Pathway as a Therapeutic Strategy in Head and Neck Squamous Cell Carcinoma.

Genomic analyses of head and neck squamous cell carcinoma (HNSCC) have highlighted alterations in the phosphatidylinositol 3-kinase (PI3K) signaling pathway, presenting a therapeutic target for multiple ongoing clinical trials with PI3K or PI3K/MTOR inhibitors. However, these inhibitors can potentially increase autophagy in HNSCC and indirectly support cancer cell survival. Here, we sought to understand the relationship between the PI3K signaling pathway and autophagy during their dual inhibition in a panel of HNSCC cell lines. We used acridine orange staining, immunoblotting, and tandem sensor Red Fluorescent Protein- Green Fluorescent Protein-, microtubule-associated protein 1 light chain 3 beta (RFP-GFP-LC3B) expression analysis to show that PI3K inhibitors increase autophagosomes in HNSCC cells, but that chloroquine treatment effectively inhibits the autophagy that is induced by PI3K inhibitors. Using the Bliss independence model, we determined that the combination of chloroquine with PI3K inhibitors works in synergy to decrease cancer cell proliferation, independent of the PIK3CA status of the cell line. Our results indicate that a strategy focusing on autophagy inhibition enhances the efficacy of therapeutics already in clinical trials. Our results suggest a broader application for this combination therapy that can be promptly translated to in vivo studies.

Non-canonical ATM/MRN activities temporally define the senescence secretory program.

Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest.

DNA Damage- But Not Enzalutamide-Induced Senescence in Prostate Cancer Promotes Senolytic Bcl-xL Inhibitor Sensitivity.

Cellular senescence is a natural tumor suppression mechanism defined by a stable proliferation arrest. In the context of cancer treatment, cancer cell therapy-induced senescence (TIS) is emerging as an omnipresent cell fate decision that can be pharmacologically targeted at the molecular level to enhance the beneficial aspects of senescence. In prostate cancer (PCa), TIS has been reported using multiple different model systems, and a more systematic analysis would be useful to identify relevant senescence manipulation molecular targets. Here we show that a spectrum of PCa senescence phenotypes can be induced by clinically relevant therapies. We found that DNA damage inducers like irradiation and poly (ADP-ribose) polymerase1 (PARP) inhibitors triggered a stable PCa-TIS independent of the p53 status. On the other hand, enzalutamide triggered a reversible senescence-like state that lacked evidence of cell death or DNA damage. Using a small senolytic drug panel, we found that senescence inducers dictated senolytic sensitivity. While Bcl-2 family anti-apoptotic inhibitor were lethal for PCa-TIS cells harboring evidence of DNA damage, they were ineffective against enzalutamide-TIS cells. Interestingly, piperlongumine, which was described as a senolytic, acted as a senomorphic to enhance enzalutamide-TIS proliferation arrest without promoting cell death. Overall, our results suggest that TIS phenotypic hallmarks need to be evaluated in a context-dependent manner because they can vary with senescence inducers, even within identical cancer cell populations. Defining this context-dependent spectrum of senescence phenotypes is key to determining subsequent molecular strategies that target senescent cancer cells.

Autophagy drives fibroblast senescence through MTORC2 regulation.

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations. Abbreviations: 3-MA: 3-methyladenine; ACTA2: actin, alpha 2, smooth muscle, aorta; AKT1: AKT serine/threonine kinase 1; p-AKT1: AKT1 Ser473 phosphorylation; t-AKT1: total AKT serine/threonine kinase 1; ATG4A: autophagy related 4A cysteine peptidase; ATG7: autophagy gene 7; C12FDG: 5-dodecanoylaminofluorescein Di-ß-D-Galactopyranoside; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; Ctl: control; DAPI: 4',6-diamidino-2-phenylindole, dilactate; ECM: extracellular matrix; GSH: L-glutathione reduced; H2O2: hydrogen peroxide; HLF: adult human lung fibroblasts; Ho: Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2.5'-bi-1H-benzimidazole); HSC: hepatic stellate cells; LY: LY294002; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTORC1/2: mechanistic target of rapamycin kinase complex 1/2; N: normal growth medium; NAC: N-acetyl-L-cysteine; PBS: phosphate-buffered saline; PDGFA: platelet derived growth factor subunit A; PRKCA/PKCα: protein kinase C alpha; PtdIns3K: class III phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; R: rapamycin; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; SA-GLB1/ß-gal: senescence-associated galactosidase beta 1; SGK1: serum/glucocorticoid regulated kinase 1; shRNA: short hairpin RNA; siCtl: control siRNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SS: serum-free (serum starvation) medium; TP53: tumor protein p53; TUBA: tubulin alpha; V: vehicle.

Single Nucleotide Polymorphism rs6942067 Is a Risk Factor in Young and in Non-Smoking Patients with HPV Negative Head and Neck Squamous Cell Carcinoma.

Genetic factors behind the increasing incidence of human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC) in young non-smokers are suspected, but have not been identified. Recently, rs6942067, a single nucleotide polymorphism (SNP) located upstream of the DCBLD1 gene, was found associated with non-smoking lung adenocarcinoma. To validate if this SNP is also implicated in HNSCC, participants of The Cancer Genome Atlas HNSCC cohort were investigated for rs6942067 status, associated DCBLD1 expression, and clinical characteristics. Occurrence of the rs6942067 GG genotype is significantly higher in young and in HPV negative non-smoking HNSCC than in other HNSCC. Additionally, rs6942067 GG is associated with higher DCBLD1 expression in HNSCC and patients with high DCBLD1 expression have a worse overall survival at three years, both in univariate and multivariate analysis. Furthermore, high DCBLD1 expression is associated with activation of the integrin signaling pathway and its phosphorylation with EGFR and MET. Collectively, these findings suggest that DCBLD1 plays a critical role in HNSCC and demonstrate an association between rs6942067 and clinical characteristics of young age and HPV negative non-smoking status in HNSCC patients.