Best practices for the dissemination and implementation of neuromuscular training injury prevention warm-ups in youth team sport: a systematic review.

OBJECTIVE: To evaluate best practices for neuromuscular training (NMT) injury prevention warm-up programme dissemination and implementation (D&I) in youth team sports, including characteristics, contextual predictors and D&I strategy effectiveness. DESIGN: Systematic review. DATA SOURCES: Seven databases were searched. ELIGIBILITY: The literature search followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. INCLUSION CRITERIA: participation in a team sport, ≥70% youth participants (<19 years), D&I outcomes with/without NMT-related D&I strategies. The risk of bias was assessed using the Downs & Black checklist. RESULTS: Of 8334 identified papers, 68 were included. Sport participants included boys, girls and coaches. Top sports were soccer, basketball and rugby. Study designs included randomised controlled trials (RCTs) (29.4%), cross-sectional (23.5%) and quasi-experimental studies (13.2%). The median Downs & Black score was 14/33. Injury prevention effectiveness (vs efficacy) was rarely (8.3%) prioritised across the RCTs evaluating NMT programmes. Two RCTs (2.9%) used Type 2/3 hybrid approaches to investigate D&I strategies. 19 studies (31.6%) used D&I frameworks/models. Top barriers were time restrictions, lack of buy-in/support and limited benefit awareness. Top facilitators were comprehensive workshops and resource accessibility. Common D&I strategies included Workshops with supplementary Resources (WR; n=24) and Workshops with Resources plus in-season Personnel support (WRP; n=14). WR (70%) and WRP (64%) were similar in potential D&I effect. WR and WRP had similar injury reduction (36-72%) with higher adherence showing greater effectiveness. CONCLUSIONS: Workshops including supplementary resources supported the success of NMT programme implementation, however, few studies examined effectiveness. High-quality D&I studies are needed to optimise the translation of NMT programmes into routine practice in youth sport.

Youth Injury Knowledge and Beliefs following Neuromuscular Training Warm-up Implementation in Schools.

Neuromuscular training warm-up programs can reduce injury rates in youth sports, but they often have poor uptake and adherence. Delivering such programs in school physical education classes may provide greater public health benefit, particularly if they promote improved injury knowledge and prevention beliefs amongst students. The purpose of this secondary analysis of a large cluster-randomized controlled trial was to understand how students' (age 11-15 years) knowledge and beliefs change after exposure to an evidence-informed neuromuscular training warm-up program. Six schools delivered the program for a 12-week period in the initial study year (n=566) and two continued to use it in a subsequent "maintenance" year (n=255). Students completed a knowledge and beliefs questionnaire at baseline, 6-week, and 12-week timepoints. Knowledge scores ranged from 7/10 to 8/10 at all timepoints and students generally believed that injuries are preventable. On average, there was less than a one-point change in knowledge between timepoints and there was no change in the median belief scores. There were no meaningful differences between sexes, grades, or previous injury. These findings highlight that knowledge and beliefs are unlikely to change passively through program exposure. More active strategies are needed to improve injury prevention perceptions in this population.

Elucidating the direct effects of the novel HDAC inhibitor bocodepsin (OKI-179) on T cells to rationally design regimens for combining with immunotherapy.

Histone deacetylase inhibitors (HDACi) are currently being explored for the treatment of both solid and hematological malignancies. Although originally thought to exert cytotoxic responses through tumor-intrinsic mechanisms by increasing expression of tumor suppressor genes, several studies have demonstrated that therapeutic responses depend on an intact adaptive immune system: particularly CD8 T cells. It is therefore critical to understand how HDACi directly affects T cells in order to rationally design regimens for combining with immunotherapy. In this study, we evaluated T cell responses to a novel class-selective HDACi (OKI-179, bocodepsin) by assessing histone acetylation levels, which revealed rapid responsiveness accompanied by an increase in CD4 and CD8 T cell frequencies in the blood. However, these rapid responses were transient, as histone acetylation and frequencies waned within 24 hours. This contrasts with in vitro models where high acetylation was sustained and continuous exposure to HDACi suppressed cytokine production. In vivo comparisons demonstrated that stopping OKI-179 treatment during PD-1 blockade was superior to continuous treatment. These findings provide novel insight into the direct effects of HDAC inhibitors on T cells and that treatment schedules that take into account acute T cell effects should be considered when combined with immunotherapies in order to fully harness the tumor-specific T cell responses in patients.

High Sport Specialization Is Associated With More Musculoskeletal Injuries in Canadian High School Students.

OBJECTIVE: To describe levels of sport specialization in Canadian high school students and investigate whether sport specialization and/or sport participation volume is associated with the history of musculoskeletal injury and/or concussion. DESIGN: Cross-sectional study. SETTING: High schools, Alberta, Canada. PARTICIPANTS: High school students (14-19 years) participating in various sports. INDEPENDENT VARIABLES: Level of sport specialization (high, moderate, low) and sport participation volume (hours per week and months per year). MAIN OUTCOME MEASURES: Twelve-month injury history (musculoskeletal and concussion). RESULTS: Of the 1504 students who completed the survey, 31% were categorized as highly specialized (7.5% before the age of 12 years). Using multivariable, negative, binomial regression (adjusted for sex, age, total yearly training hours, and clustering by school), highly specialized students had a significantly higher musculoskeletal injury rate [incidence rate ratio (IRR) = 1.36, 95% confidence interval (CI), 1.07-1.73] but not lower extremity injury or concussion rate, compared with low specialization students. Participating in one sport for more than 8 months of the year significantly increased the musculoskeletal injury rate (IRR = 1.27, 95% CI, 1.02-1.58). Increased training hours significantly increased the musculoskeletal injury rate (IRR = 1.18, 95% CI, 1.13-1.25), lower extremity injury rate (IRR = 1.16, 95% CI, 1.09-1.24), and concussion rate (IRR = 1.31, 95% CI, 1.24-1.39). CONCLUSIONS: Approximately one-third of Canadian high school students playing sports were categorized as highly specialized. The musculoskeletal injury rate was higher for high sport specialization students compared with low sport specialization students. Musculoskeletal injuries and concussion were also more common in students who train more and spend greater than 8 months per year in one sport.

Who Does Not Respond to Injury Prevention Warm-up Programs? A Secondary Analysis of Trial Data From Neuromuscular Training Programs in Youth Basketball, Soccer, and Physical Education.

OBJECTIVES: To identify factors associated with nonresponse to neuromuscular training (NMT) warm-up programs among youth exposed to NMT warm-ups. METHODS: This is a secondary analysis of youth (aged 11-18 years) in the intervention groups of 4 randomized controlled trials in high school basketball, youth community soccer, and junior high school physical education. Youth who were exposed to NMT and who sustained an injury during the study were considered nonresponders. Odds ratios (ORs) were based on generalized estimating equations logistic regression controlling for clustering by team/class and adjusted for age, weight, height, balance performance, injury history, sex, and sport (soccer/basketball/physical education). RESULTS: A total of 1793 youth were included. Youth with a history of injury in the previous year had higher odds (OR = 1.64; 95% CI: 1.14, 2.37) of injury during the study, and females were more likely (OR = 1.67; 95% CI: 1.21, 2.31) to sustain an injury than males who were participating in NMT. Age was not associated with the odds of sustaining an injury (OR = 1.10; 95% CI: 0.93, 1.30). Soccer players benefited most from greater adherence, with 81% lower odds of injury (OR = 0.19; 95% CI: 0.06, 0.57) when completing 3 NMT sessions a week compared with 1 session per week. CONCLUSION: Factors associated with nonresponse to an NMT warm-up program were female sex, history of injury during the previous 12 months, and lower weekly NMT session adherence in some sports (soccer). J Orthop Sports Phys Ther 2023;53(2):94-102. Epub: 9 December 2022. doi:10.2519/jospt.2022.11526.

Covalent JNK Inhibitor, JNK-IN-8, Suppresses Tumor Growth in Triple-Negative Breast Cancer by Activating TFEB- and TFE3-Mediated Lysosome Biogenesis and Autophagy.

The heterogeneity and aggressiveness of triple-negative breast cancer (TNBC) contribute to its early recurrence and metastasis. Despite substantial research to identify effective therapeutic targets, TNBC remains elusive in terms of improving patient outcomes. Here, we report that a covalent JNK inhibitor, JNK-IN-8, suppresses TNBC growth both in vitro and in vivo. JNK-IN-8 reduced colony formation, cell viability, and organoid growth in vitro and slowed patient-derived xenograft and syngeneic tumor growth in vivo. Cells treated with JNK-IN-8 exhibited large, cytoplasmic vacuoles with lysosomal markers. To examine the molecular mechanism of this phenotype, we looked at the master regulators of lysosome biogenesis and autophagy transcription factor EB (TFEB) and TFE3. JNK-IN-8 inhibited TFEB phosphorylation and induced nuclear translocation of unphosphorylated TFEB and TFE3. This was accompanied by an upregulation of TFEB/TFE3 target genes associated with lysosome biogenesis and autophagy. Depletion of both TFEB and TFE3 diminished the JNK-IN-8-driven upregulation of lysosome biogenesis and/or autophagy markers. TFEB and TFE3 are phosphorylated by a number of kinases, including mTOR. JNK-IN-8 reduced phosphorylation of mTOR targets in a concentration-dependent manner. Knockout of JNK1 and/or JNK2 had no impact on TFEB/TFE3 activation or mTOR inhibition by JNK-IN-8 but inhibited colony formation. Similarly, reexpression of either wildtype or drug-nonbinding JNK (C116S) in JNK knockout cells did not reverse JNK-IN-8-induced TFEB dephosphorylation. In summary, JNK-IN-8 induced lysosome biogenesis and autophagy by activating TFEB/TFE3 via mTOR inhibition independently of JNK. Together, these findings demonstrate the efficacy of JNK-IN-8 as a targeted therapy for TNBC and reveal its novel lysosome- and autophagy-mediated mechanism of action.

Sport Specialization, Physical Performance and Injury History in Canadian Junior High School Students.

BACKGROUND: Youth sports participation is encouraged for proposed physical and psychological benefits. However early sport specialization and the potentially negative consequences may be a cause for concern. PURPOSE: To describe sport specialization in Canadian youth and investigate associations with previous injury and physical performance. STUDY DESIGN: Cross-sectional study. METHODS: Junior high school students (grades 7-9, ages 11-16) were invited to participate. All participants completed a questionnaire capturing specialization level (low, moderate, high; based on year-round training, exclusion of other sports, and single-sport training) and injury history in the previous 12-months. Additionally, all participants completed physical performance measures including vertical jump (cm), predicted VO2max (mL/kg/min), single-leg balance (secs) and Y-Balance composite score (%). Logistic regression examined the association between school grade, school size, sex and sport specialization (Objective 1) and the association between sport specialization and injury history (Objective 2). Multivariable linear regression analyses (4) assessed associations between sport specialization category and physical performance measures (Objective 3). RESULTS: Two hundred and thirty-eight students participated in the study. Eighteen percent of participants reported high specialization, with no significant associations between sex, grade or school size and specialization category. There was no significant difference in the odds of sustaining previous injury between participants reporting moderate (odds ratio [OR]=1.94, 95% CI 0.86-4.35) or high (OR=2.21, 95% CI 0.43-11.37) compared to low specialization. There were no significant differences in vertical jump height (mean diff [MD] = -0.4 to 2.1cm), predicted VO2max (MD = 2.2 to 3.1mL/kg/min), single leg balance (MD = 0.5 to 1.9sec) or Y-balance (MD = 0.6 to 7.0%) between sport specialization categories. CONCLUSIONS: Sport specialization exists in Canadian junior high schools but may be less common than previously reported and it was not associated with sex, grade, or school size. Level of specialization was not associated with history of injury nor a range of physical performance measures. LEVEL OF EVIDENCE: Level 3.

Canadian High School Rugby Coaches Readiness for an Injury Prevention Strategy Implementation: Evaluating a Train-the-Coach Workshop.

Background: Canadian rugby coach injury prevention beliefs and attitudes have not been studied, yet are key to informing injury prevention strategy implementation. Despite neuromuscular training (NMT) warm-up success in reducing injury, adoption of these programs is variable. Therefore, objectives of this study included (1) describing Canadian youth rugby coach injury prevention beliefs and attitudes and current warm-up practices and (2) evaluating intention to use a rugby-specific NMT warm-up. Methods: High school rugby coaches completed a questionnaire before and after a rugby-specific NMT warm-up workshop. The pre-workshop questionnaire captured demographics, current warm-up practice, and NMT warm-up knowledge and use. Both questionnaires captured injury prevention beliefs, attitudes and behavioral intention. Results: Forty-eight coaches participated in the workshops. Pre-workshop, 27% of coaches were aware of NMT warm-ups. Coaches primarily included aerobic and stretching components, while balance components were not common in their warm-ups over the past year. Additionally, 92% of coaches agreed to some extent they would "complete a rugby-specific warm-up program prior to every game and training session this season." Post-workshop, 86% of coaches agreed to some extent that they would use the program in every rugby session. No differences were observed between pre- and post-workshop intention to implement the warm-up (p = 0.10). Interpretation: This is the first study to examine current Canadian youth rugby coach warm-up practices and intention to use NMT warm-ups. Canadian rugby coach intention to use a rugby-specific NMT warm-up is high, providing ample opportunity to investigate the efficacy of a NMT warm-up in youth rugby.

BCDIN3D RNA methyltransferase stimulates Aldolase C expression and glycolysis through let-7 microRNA in breast cancer cells.

Type II diabetes (T2D) and specific cancers share many risk factors, however, the molecular mechanisms underlying these connections are often not well-understood. BCDIN3D is an RNA modifying enzyme that methylates specific precursor microRNAs and tRNAHis. In addition to breast cancer, BCDIN3D may also be linked to metabolism, as its gene locus is associated with obesity and T2D. In order to uncover metabolic pathways regulated by BCDIN3D in cancer, we performed an unbiased analysis of the metabolome, transcriptome, and proteome of breast cancer cells depleted for BCDIN3D. Intersection of these analyses showed that BCDIN3D-depleted cells have increased levels of Fructose 1,6 Bisphosphate (F1,6-BP), the last six-carbon glycolytic intermediate accompanied by reduced glycolytic capacity. We further show that elevated F1,6-BP is due to downregulation of Aldolase C (ALDOC), an enzyme that cleaves F1,6-BP mainly in the brain, but whose high expression/amplification is associated with poor prognosis in breast cancer. BCDIN3D regulates ALDOC through a non-canonical mechanism involving the crucial let-7 microRNA family and its target site on the 3'UTR of ALDOC. Overall, our results reveal an important connection between BCDIN3D, let-7 and glycolysis that may be relevant to breast cancer, obesity, and T2D.

Warm-Ups and Coaches' Perceptions: Searching for Clues to Improve Injury Prevention in Youth Basketball.

Introduction: Regular use of neuromuscular training (NMT) warm-up programs improves performance and prevents injuries. However, low level of adoption of these programs remains a problem. Understanding the current warm-ups in youth basketball and coaches' perceptions on injury prevention can guide the design of superior implementation strategies. This study describes warm-ups in youth basketball and coaches' injury prevention-related knowledge, attitudes, beliefs, and information sources. Methods: Youth basketball coaches (n = 50) completed a preseason questionnaire. The questionnaire covered warm-up length, use of aerobic/agility/balance/strength/other exercises in the warm-up, injury-related knowledge, attitudes, beliefs, and sources of information. Results: Typical warm-up duration was ≤ 10 min (48.0% of coaches, 95% CI: ±13.8%). All coaches included aerobic exercises in their warm-up. Agility, strength, and balance exercises were utilized by 80.0% (95% CI: ±11.7%), 70.7% (95% CI: ±13.6%), and 26.8% (95% CI: ±13.6%) of coaches, respectively. Most coaches agreed to some extent that basketball injuries are preventable (94%) and that participating in a NMT warm-up program would reduce player's risk of injury (92%). Other coaches were identified as the most common source of information on warm-ups and injury prevention. Discussion: Coaches use parts of effective NMT warm-up programs, but balance exercises are not well adopted. Considering the level of evidence supporting the importance of balance exercises in injury prevention, it is crucial to improve the implementation of NMT warm-up programs in youth basketball, for example, through educational courses. As fellow coaches were identified as the most important source of information, coaches' role in knowledge translation should be emphasized.

Facilitators and Barriers to the Implementation of iSPRINT: A Sport Injury Prevention Program in Junior High Schools.

OBJECTIVES: Sport injury is the leading cause of hospitalization in Canadian youth and represents a high burden to the health care system. This study aims to describe the facilitators and barriers to implementation of a sport injury prevention program in junior high school physical education (known as iSPRINT), previously shown to reduce the risk of sport-related injury in youth (age, 11-15 years). METHODS: Focus group data were mapped onto constructs from the Consolidated Framework for Implementation Research (CFIR). Four schools that implemented iSPRINT participated in this study. Forty-seven key stakeholders (teachers, students, principals) participated in 9 semistructured focus groups and 4 interviews. The CFIR was used to guide the focus group discussions, data coding, and analysis using a qualitative content analysis approach. RESULTS: Of the 22 applicable CFIR constructs, 16 were identified in the transcripts. The most significant facilitators to successful implementation efforts included evidence strength and quality, adaptability, implementation climate, culture, and having a high level of compatibility facilitated successful implementation efforts. Barriers to implementation included intervention complexity, planning, and readiness for implementation. Constructs that acted as both a facilitator and a barrier, depending on the context, were self-efficacy, execution, and individual identification with the organization. CONCLUSIONS: Participants in this study reported positive attitudes about implementing iSPRINT, citing evidence strength, adaptability, and constructs related to the organizational setting that contributed to successful implementation. Potential improvements include modifying certain program components, decreasing the number of components, and reducing the equipment required.

Implementing a junior high school-based programme to reduce sports injuries through neuromuscular training (iSPRINT): a cluster randomised controlled trial (RCT).

OBJECTIVE: To evaluate the effectiveness of a junior high school-based sports injury prevention programme to reduce injuries through neuromuscular training (NMT). METHODS: This was a cluster randomised controlled trial. Students were recruited from 12 Calgary junior high schools (2014-2017). iSPRINT is a 15 min NMT warm-up including aerobic, agility, strength and balance exercises. Following a workshop, teachers delivered a 12-week iSPRINT NMT (six schools) or a standard-of-practice warm-up (six schools) in physical education classes. The definition of all recorded injuries included injuries that resulted in participants being unable to complete a sport and recreation (S&R) session, lost time from sport and/or seek medical attention. Incidence rate ratios (IRRs) were estimated based on multiple multilevel Poisson regression analyses (adjusting for sex (considering effect modification) and previous injury, offset by S&R participation hours, and school-level and class-level random effects were examined) for intent-to-treat analyses. RESULTS: 1067 students (aged 11-16) were recruited across 12 schools (6 intervention schools (22 classes), 6 control schools (27 classes); 53.7% female, 46.3% male). The iSPRINT programme was protective of all recorded S&R injuries for girls (IRR=0.543, 95% CI 0.295 to 0.998), but not for boys (IRR=0.866, 95% CI 0.425 to 1.766). The iSPRINT programme was also protective of each of lower extremity injuries (IRR=0.357, 95% CI 0.159 to 0.799) and medical attention injuries (IRR=0.289, 95% CI 0.135 to 0.619) for girls, but not for boys (IRR=1.055, 95% CI 0.404 to 2.753 and IRR=0.639, 95% CI 0.266 to 1.532, respectively). CONCLUSION: The iSPRINT NMT warm-up was effective in preventing each of all recorded injuries, lower extremity injuries and medically treated S&R injuries in female junior high school students. TRIAL REGISTRATION NUMBER: NCT03312504.

Single-cell RNA sequencing of lung adenocarcinoma reveals heterogeneity of immune response-related genes.

Immunotherapy has emerged as a promising approach to treat cancer. However, partial responses across multiple clinical trials support the significance of characterizing intertumor and intratumor heterogeneity to achieve better clinical results and as potential tools in selecting patients for different types of cancer immunotherapies. Yet, the type of heterogeneity that informs clinical outcome and patient selection has not been fully explored. In particular, the lack of characterization of immune response-related genes in cancer cells hinders the further development of metrics to select and optimize immunotherapy. Therefore, we analyzed single-cell RNA-Seq data from lung adenocarcinoma patients and cell lines to characterize the intratumor heterogeneity of immune response-related genes and demonstrated their potential impact on the efficacy of immunotherapy. We discovered that IFN-γ signaling pathway genes are heterogeneously expressed and coregulated with other genes in single cancer cells, including MHC class II (MHCII) genes. The downregulation of genes in IFN-γ signaling pathways in cell lines corresponds to an acquired resistance phenotype. Moreover, analysis of 2 groups of tumor-restricted antigens, namely neoantigens and cancer testis antigens, revealed heterogeneity in their expression in single cells. These analyses provide a rationale for applying multiantigen combinatorial therapies to prevent tumor escape and establish a basis for future development of prognostic metrics based on intratumor heterogeneity.

A c-Jun N-terminal kinase inhibitor, JNK-IN-8, sensitizes triple negative breast cancer cells to lapatinib.

Triple negative breast cancers (TNBC) have poor prognosis compared to other breast cancer subtypes and represent 15-20% of breast cancers diagnosed. Unique targets and new molecularly-targeted therapies are urgently needed for this subtype. Despite high expression of Epidermal Growth Factor Receptor, inhibitors such as lapatinib have not shown therapeutic efficacy in TNBC patients. Herein, we report that treatment with the covalent JNK inhibitor, JNK-IN-8, synergizes with lapatinib to cause cell death, while these compounds as single agents have little effect. The combination significantly increases survival of mice bearing xenografts of MDA-MB-231 human TNBC cells. Our studies demonstrate that lapatinib treatment increases c-Jun and JNK phosphorylation indicating a mechanism of resistance. Combined, these compounds significantly reduce transcriptional activity of Nuclear Factor kappa B, Activating Protein 1, and Nuclear factor erythroid 2-Related Factor 2. As master regulators of antioxidant response, their decreased activity induces a 10-fold increase in reactive oxygen species that is cytotoxic, and is rescued by addition of exogenous antioxidants. Over expression of p65 or Nrf2 also significantly rescues viability during JNK-IN-8 and lapatinib treatment. Further studies combining JNK-IN-8 and lapatinib may reveal a benefit for patients with TNBC, fulfilling a critical medical need.

Serotonin Analogues as Inhibitors of Breast Cancer Cell Growth.

Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.

Extracellular Matrix Stiffening Induces a Malignant Phenotypic Transition in Breast Epithelial Cells.

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established in vitro models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator. Non-malignant MCF10A mammary epithelial cells (MECs) lose their epithelial characteristics and become invasive when cultured in stiff microenvironments, leading to the hypothesis that tumor stiffening could contribute directly to disease progression. However, previous studies demonstrating MCF10A invasion have been performed in gels with constant mechanical properties, unlike the dynamically stiffening tumor microenvironment. Here, we employ a temporally stiffening hydrogel platform to demonstrate that matrix stiffening induces invasion from and proliferation in MCF10A mammary acini. After allowing MCF10A acini to form in soft hydrogels for 14 days, the gels were stiffened to the level of a malignant tumor, giving rise to a proliferative and invasive phenotype. Cells were observed to collectively migrate away from mammary acini while maintaining cell-cell contacts. Small molecule inhibition of PI3K and Rac1 pathways was sufficient to significantly reduce the number and size of invasive acini after stiffening. Our results demonstrate that temporal matrix stiffening can induce invasion from mammary acini and supports the notion that tumor stiffening could be implicated in disease progression and metastasis.

ATM regulation of IL-8 links oxidative stress to cancer cell migration and invasion.

Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression.

c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

c-Jun N-Terminal Kinases Mediate a Wide Range of Targets in the Metastatic Cascade.

Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade.

c-Jun N-terminal Kinase 2 Regulates Multiple Receptor Tyrosine Kinase Pathways in Mouse Mammary Tumor Growth and Metastasis.

c-Jun N-terminal kinase 2 (JNK2) isoforms are transcribed from the jnk2 gene and are highly homologous with jnk1 and jnk3 transcriptional products. JNK proteins mediate cell proliferation, stress response, and migration when activated by a variety of stimuli, including receptor tyrosine kinases (RTKs), but their ability to influence tumor metastasis is ill defined. To evaluate JNK2 in this manner, we used the highly metastatic 4T1.2 mammary tumor cells. Short hairpin RNA expression directed toward JNK2 (shJNK2) decreases tumor cell invasion. In vivo, shJNK2 expression slows tumor growth and inhibits lung metastasis. Subsequent analysis of tumors showed that shJNK2 tumors express lower GRB2-associated binding protein 2 (GAB2). In vitro, knockdown of JNK2 or GAB2 inhibits Akt activation by hepatocyte growth factor (HGF), insulin, and heregulin-1, while phosphorylation of ERK is constitutive and Src dependent. Knockdown of GAB2 phenocopies knockdown of JNK2 in vivo by reducing tumor growth and metastasis, supporting that JNK2 mediates tumor progression by regulating GAB2. The influence of jnk2 in the host or microenvironment was also evaluated using syngeneic jnk2-/- and jnk2+/+ mice. Jnk2-/- mice experience longer survival and less bone and lung metastasis compared to jnk2+/+ mice after intracardiac injection of 4T1.2 cells. GAB2 has previously been shown to mediate osteoclast differentiation, and osteoclasts are critical mediators of tumor-related osteolysis. Thus, studies focusing on the role of JNK2 on osteoclast differentiation were undertaken. ShJNK2 expression impairs osteoclast differentiation, independently of GAB2. Further, shJNK2 4T1.2 cells express less RANKL, a stimulant of osteoclast differentiation. Together, our data support that JNK2 conveys Src/phosphotidylinositol 3-kinase (PI3K) signals important for tumor growth and metastasis by enhancing GAB2 expression. In osteoclast progenitor cells, JNK2 promotes differentiation, which may contribute to the progression of bone metastasis. These studies identify JNK2 as a tumor and host target to inhibit breast cancer growth and metastasis.