Posttranscriptional control of T cell effector function by aerobic glycolysis.

A "switch" from oxidative phosphorylation (OXPHOS) to aerobic glycolysis is a hallmark of T cell activation and is thought to be required to meet the metabolic demands of proliferation. However, why proliferating cells adopt this less efficient metabolism, especially in an oxygen-replete environment, remains incompletely understood. We show here that aerobic glycolysis is specifically required for effector function in T cells but that this pathway is not necessary for proliferation or survival. When activated T cells are provided with costimulation and growth factors but are blocked from engaging glycolysis, their ability to produce IFN-γ is markedly compromised. This defect is translational and is regulated by the binding of the glycolysis enzyme GAPDH to AU-rich elements within the 3' UTR of IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through fluctuations in its expression, controls effector cytokine production. Thus, aerobic glycolysis is a metabolically regulated signaling mechanism needed to control cellular function.

Assessment of Ground Contact Time in the Field: Evaluation of Validity and Reliability.

ABSTRACT: Weber, JA, Hart, NH, Rantalainen, T, Connick, M, and Newton, RU. Assessment of ground contact time in the field: evaluation of validity and reliability. J Strength Cond Res 38(1): e34-e39, 2024-The capacity to measure the kinetic and kinematic components of running has been extensively investigated in laboratory settings. Many authors have produced work that is of high value to practitioners within sporting environments; however, the lack of field-based technology to assess features of running gait validly and reliably has prevented the application of these valuable works. This paper examines the validity and reliability of a practical field-based methodology for using commercial inertial measurement units (IMUs) to assess ground contact time (GCT). Validity was examined in the comparison of GCT measured from ground reaction force by a force plate and that determined by a lumbar mounted commercial IMU and analyzed using a commercially available system (SPEEDSIG). Reliability was assessed by a field-based examination of within and between-session variability in GCT measured using a commercially available system (SPEEDSIG). Significance was set at p ≤ 0.05. Results for validity (intraclass correlation [ICC] 0.83) and reliability (ICC 0.91) confirm that the described field-based methodology is qualified for use to determine GCT in a practical setting. The implications of this study are important as they offer sport practitioners (S&C coaches, rehab specialists, and physios) a scalable method to assess GCT in the field to develop greater understanding of their athletes and improve performance, injury prevention, and rehabilitation interventions. Furthermore, these results provide the foundation for further work that could provide greater detail describing individual running gait in the field.

Relationship Between Mechanical Effectiveness in Sprint Running and Force-Velocity Characteristics of a Countermovement Jump in Australian Rules Football Athletes.

ABSTRACT: Morris, CG, Weber, JA, and Netto, KJ. Relationship between mechanical effectiveness in sprint running and force-velocity characteristics of a countermovement jump in Australian rules football athletes. J Strength Cond Res 36(3): e59-e65, 2022-This study evaluated the mechanical determinants of 40-m sprint performance in elite Australian Rules Football (ARF) athletes and identified variables of countermovement jumps (CMJs) that related to the sprint. Fourteen elite male ARF athletes (age = 22.7 ± 3.6 years; height = 1.88 ± 0.08 m; mass = 88.2 ± 9.38 kg) completed two 40-m sprints and 3 CMJs. Sprint mechanics were calculated using inverse dynamic methods from sprint times, anthropometric and spatiotemporal data, whereas CMJ variables were obtained from in-ground force plates. Associations between sprint mechanics, sprint performance, and CMJ variables were identified using Pearson's correlation coefficient. A p-value of <0.036 was considered statistically significant for all analyses after performing Bonferroni correction adjustment. Relative peak running power was significantly correlated (p < 0.036, r = -0.781 to -0.983) with sprint split times across all distances (5-40 m). Relative maximum horizontal force significantly correlated with acceleration performance (0-20 m, p < 0.036, r = -0.887 to -0.989). Maximum running velocity was significantly correlated (p < 0.036, r = -0.714 to -0.970) with sprint times across 20-40 m. Relative peak force in the CMJ was significantly associated (p < 0.036, r = -0.589 to -0.630) with sprint kinetics (power and horizontal force) and 5-20-m sprint times. Jump height and concentric time in the CMJ were significantly (p < 0.036) correlated with sprint time at 20 m (r = -0.550 and r = 0.546), respectively. These results indicate emphasis should be placed on training protocols that improve relative peak power, particularly in time-constrained environments such as team sports, focusing on maximal force production or maximal running velocity ability. Furthermore, associations between CMJ variables and sprint performance provide practitioners with an approach to assess sprint performance in-season, monitor training adaptations and further individualize training interventions, without requiring maximal sprint testing.

Functional Basis of Asymmetrical Lower-Body Skeletal Morphology in Professional Australian Rules Footballers.

Hart, NH, Newton, RU, Weber, J, Spiteri, T, Rantalainen, T, Dobbin, M, Chivers, P, and Nimphius, S. Functional basis of asymmetrical lower-body skeletal morphology in elite Australian footballers. J Strength Cond Res 34(3): 791-799, 2020-Bone strength is a product of its material and structural properties and is highly responsive to mechanical load. Given the measureable and adaptable features of bone, and thus relevance to medical screening, injury prevention, and injury management in athletes, this study describes the lower-body skeletal morphology of professional Australian rules footballers. Using a cross-sectional and quantitative study design, 54 professional Australian rules football players (n = 54; age: 22.4 ± 3.8 years; height: 189.0 ± 7.5 cm; body mass: 86.0 ± 8.6 kg; tibial length: 436.1 ± 29.2 mm; and body fat: 9.9 ± 1.7%) underwent tibiofibular peripheral quantitative computed tomography scans for the kicking and support limbs, and a whole-body dual-energy X-ray absorptiometry scans. The support leg was significantly stronger than the kicking leg (bone strength: p ≤ 0.001; d = 0.47) with significantly greater bone mass (p < 0.001; d = 0.28), cross-sectional areas (p ≤ 0.002; d = 0.20), and greater cortex thickness (p = 0.017; d = 0.20), owing to significantly greater periosteal apposition (p ≤ 0.001; d = 0.29) and endocortical expansion (p = 0.019; d = 0.13), despite significantly lower cortical density (p = 0.002; d = -0.25). Disparate skeletal morphology between limbs highlights context-specific adaptive responses to mechanical loads experienced during game-based tasks. Practitioners should concomitantly measure material and structural properties of musculoskeletal tissue when examining fragility or resilience to better inform medical screening, monitoring, and injury risk stratification. Support leg axial loading highlights a potential avenue for interventions aiming to remediate or optimize bone cross-sectional area.

Assessment of a Novel Algorithm to Determine Change-of-Direction Angles While Running Using Inertial Sensors.

Balloch, AS, Meghji, M, Newton, RU, Hart, NH, Weber, JA, Ahmad, I, and Habibi, D. Assessment of a novel algorithm to determine change-of-direction angles while running using inertial sensors. J Strength Cond Res 34(1): 134-144, 2020-The ability to detect and quantify change-of-direction (COD) movement may offer a unique approach to load-monitoring practice. Validity and reliability of a novel algorithm to calculate COD angles for predetermined COD movements ranging from 45 to 180° in left and right directions was assessed. Five recreationally active men (age: 29.0 ± 0.5 years; height: 181.0 ± 5.6 cm; and body mass: 79.4 ± 5.3 kg) ran 5 consecutive predetermined COD trials each, at 4 different angles (45, 90, 135, and 180°), in each direction. Participants were fitted with a commercially available microtechnology unit where inertial sensor data were extracted and processed using a novel algorithm designed to calculate precise COD angles for direct comparison with a high-speed video (remotely piloted, position-locked aircraft) criterion measure. Validity was assessed using Bland-Altman 95% limits of agreement and mean bias. Reliability was assessed using typical error (expressed as a coefficient of variation [CV]). Concurrent validity was present for most angles. Left: (45° = 43.8 ± 2.0°; 90° = 88.1 ± 2.0°; 135° = 136.3 ± 2.1°; and 180° = 181.8 ± 2.5°) and Right: (45° = 46.3 ± 1.6°; 90° = 91.9 ± 2.2°; 135° = 133.4 ± 2.0°; 180° = 179.2 ± 5.9°). All angles displayed excellent reliability (CV < 5%) while greater mean bias (3.6 ± 5.1°, p < 0.001), weaker limits of agreement, and reduced precision were evident for 180° trials when compared with all other angles. High-level accuracy and reliability when detecting COD angles further advocates the use of inertial sensors to quantify sports-specific movement patterns.

Associations of race and ethnicity with risk of developing invasive breast cancer after lobular carcinoma in situ.

BACKGROUND: Lobular carcinoma in situ (LCIS) of the breast is a risk factor of developing invasive breast cancer. We evaluated the racial differences in the risks of subsequent invasive breast cancer following LCIS. METHODS: We utilized data from the Surveillance, Epidemiology, and End Results registries to identify 18,835 women diagnosed with LCIS from 1990 to 2015. Cox proportional hazards regression was used to estimate race/ethnicity-associated hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of subsequent invasive breast cancer. RESULTS: During a median follow-up of 90 months, 1567 patients developed invasive breast cancer. The 10-year incidence was 7.9% for Asians, 8.2% for Hispanics, 9.3% for whites, and 11.2% for blacks (P = 0.046). Compared to white women, black women had significantly elevated risks of subsequent invasive breast cancer (HR 1.33; 95% CI 1.11, 1.59), and invasive cancer in the ipsilateral breast (HR 1.37; 95% CI 1.08, 1.72) and in the contralateral breast (HR 1.33; 95% CI 1.00, 1.76). Black women had significantly higher risks of invasive subtypes negative for both estrogen receptor and progesterone receptor (HR 1.86; 95% CI 1.14, 3.03) and invasive subtypes positive for one or both of receptors (HR 1.30; 95% CI 1.07, 1.59). The risk of subsequent invasive breast cancer was comparable in Asian women and Hispanic women compared with white women. CONCLUSIONS: Black women had a significantly higher risk of developing invasive breast cancer, including both hormone receptor-positive and hormone receptor-negative subtypes, after LCIS compared with white counterparts. It provides an opportunity to address health disparities.

Race and risk of subsequent aggressive breast cancer following ductal carcinoma in situ.

BACKGROUND: General populations of black women have a higher risk of developing breast cancer negative for both estrogen receptor (ER) and progesterone receptor (PR) in comparison with white counterparts. Racial differences remain unknown in the risk of developing aggressive invasive breast cancer (IBC) that is characterized by negativity for both ER and PR (ER-PR-) or higher 21-gene recurrence scores after ductal carcinoma in situ (DCIS). METHODS: This study identified 163,892 women (10.5% black, 9.8% Asian, and 8.6% Hispanic) with incident DCIS between 1990 and 2015 from the Surveillance, Epidemiology, and End Results data sets. Cox proportional hazards regression was used to estimate hazards ratios (HRs) of subsequent IBC classified by the hormone receptor status and 21-gene recurrence scores. RESULTS: During a median follow-up of 90 months, 8333 women developed IBC. In comparison with white women, the adjusted HR of subsequent ER-PR- breast cancer was 1.86 (95% confidence interval [CI], 1.57-2.20) for black women (absolute 10-year difference, 2.2%) and 1.40 (95% CI, 1.14-1.71) for Asian women (absolute 10-year difference, 0.4%); this was stronger than the associations for ER+ and/or PR+ subtypes (Pheterogeneity  = .0004). The 21-gene recurrence scores of subsequent early-stage, ER+ IBCs varied by race/ethnicity (Pheterogeneity  = .057); black women were more likely than white women to have a recurrence score of 26 or higher (HR, 1.38; 95% CI, 1.00-1.92). No significant difference was observed in the risks of subsequent IBC subtypes for Hispanic women. CONCLUSIONS: Black and Asian women with DCIS had higher risks of developing biologically aggressive IBC than white counterparts. This should be considered in treatment decisions for black and Asian patients with DCIS.

Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Overexpression of the multiple myeloma set domain (MMSET) Wolf-Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t(4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138+ tumor cells from t(4;14)-positive and -negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t(4;14)-negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11-driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid-derived 2)-like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11-driven increases in ROS; however, transcriptional up-regulation of some of NRF2's antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.-Mahajan, N., Wu, H.-J., Bennett, R. L., Troche, C., Licht, J. D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Whole-genome analysis informs breast cancer response to aromatase inhibition.

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

ARF tumor suppression in the nucleolus.

Since its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

Targeting PTEN-defined breast cancers with a one-two punch.

With tremendous advances in sequencing and analysis in recent years, a wealth of genetic information has become available to identify and classify breast cancer into five main subtypes - luminal A, luminal B, claudin-low, human epidermal growth factor receptor 2-enriched, and basal-like. Current treatment decisions are often based on these classifications, and while more beneficial than any single treatment for all breast cancers, targeted therapeutics have exhibited limited success with most of the subtypes. Luminal B breast cancers are associated with early relapse following endocrine therapy and often exhibit a poor prognosis that is similar to that of the aggressive basal-like breast cancers. Identifying genetic components that contribute to the luminal B endocrine resistant phenotype has become imperative. To this end, numerous groups have identified activation of the phosphatidylinositol 3-kinase (PI3K) pathway as a common recurring event in luminal B cancers with poor outcome. Examining the pathways downstream of PI3K, Fu and colleagues have recreated a human model of the luminal B subtype of breast cancer. The authors were able to reduce expression of phosphatase and tensin homolog (PTEN), the negative regulator of PI3K, using inducible short hairpin RNAs. By varying the expression of PTEN, the authors effectively conferred endocrine resistance and recapitulated the luminal B gene expression signature. Using this system in vitro and in vivo, they then tested the ability of selective kinase inhibitors downstream of PI3K to enhance current endocrine therapies. A combination of fulvestrant, which blocks ligand-dependent and -independent estrogen receptor signaling, with protein kinase B inhibition was found to overcome endocrine resistance. These findings squarely place PTEN expression levels at the nexus of luminal B breast cancers and indicates that patients with PTEN-low estrogen receptor-positive tumors might benefit from combined endocrine and PI3K pathway therapies.

Elevated DDX21 regulates c-Jun activity and rRNA processing in human breast cancers.

INTRODUCTION: The DDX21 RNA helicase has been shown to be a nucleolar and nuclear protein involved in ribosome RNA processing and AP-1 transcription. DDX21 is highly expressed in colon cancer, lymphomas, and some breast cancers, but little is known about how DDX21 might promote tumorigenesis. METHODS: Immunohistochemistry was performed on a breast cancer tissue array of 187 patients. In order to study the subcellular localization of DDX21 in both tumor tissue and tumor cell lines, indirect immunofluorescence was applied. The effect of DDX21 knockdown was measured by cellular apoptosis, rRNA processing assays, soft agar growth and mouse xenograft imaging. AP-1 transcriptional activity was analyzed with a luciferase reporter and bioluminescence imaging, as well as qRT-PCR analysis of downstream target, cyclin D1, to determine the mechanism of action for DDX21 in breast tumorigenesis. RESULTS: Herein, we show that DDX21 is highly expressed in breast cancer tissues and established cell lines. A significant number of mammary tumor tissues and established breast cancer cell lines exhibit nuclear but not nucleolar localization of DDX21. The protein expression level of DDX21 correlates with cell proliferation rate and is markedly induced by EGF signaling. Mechanistically, DDX21 is required for the phosphorylation of c-Jun on Ser73 and DDX21 deficiency markedly reduces the transcriptional activity of AP-1. Additionally, DDX21 promotes rRNA processing in multiple breast cancer cell lines. Tumor cells expressing high levels of endogenous DDX21 undergo apoptosis after acute DDX21 knockdown, resulting in significant reduction of tumorigenicity in vitro and in vivo. CONCLUSIONS: Our findings indicate that DDX21 expression in breast cancer cells can promote AP-1 activity and rRNA processing, and thus, promote tumorigenesis by two independent mechanisms. DDX21 could serve as a marker for a subset of breast cancer patients with higher proliferation potential and may be used as a therapeutic target for a subset of breast cancer patients.

Activation of PKR by a short-hairpin RNA.

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

Activation of PKR by a short-hairpin RNA.

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells.

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation.

Induction of viral mimicry upon loss of DHX9 and ADAR1 in breast cancer cells.

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The interferon inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for ADAR1 in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110 interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways.

Stromal and therapy-induced macrophage proliferation promotes PDAC progression and susceptibility to innate immunotherapy.

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

Nucleolar disruption ensures nuclear accumulation of p21 upon DNA damage.

p21(cip1) is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21(cip1) increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21(cip1) is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21(cip1) not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady-state distribution of p21(cip1) in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21(cip1) and correlated with the inhibition of p21(cip1) nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant-negative mutant of nucleophosmin induced p21(cip1) accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21(cip1) in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21(cip1).

Knocking down nucleolin expression in gliomas inhibits tumor growth and induces cell cycle arrest.

Nucleolin is a multifunctional protein whose expression often correlates with increased cellular proliferation. While the expression of nucleolin is often elevated in numerous cancers, its expression in normal human brain and in astrocytomas has not been previously reported. Using paraffin-embedded sections from normal adult autopsy specimens and glioma resection specimens, we demonstrate that nucleolin expression is limited in the normal human brain specifically to mature neurons, ependymal cells, and granular cells of the dentate gyrus. While astrocytes in the normal human brain do not express nucleolin at significant levels, glioblastoma cell lines and primary human astrocytoma cells exhibit considerable nucleolin expression. Reduction of nucleolin expression through siRNA-mediated knockdown in the U87MG glioblastoma cell line caused a dramatic decrease in cell proliferation and induced cell cycle arrest in vitro. Moreover, conditional siRNA knockdown of nucleolin expression in U87MG intracranial xenografts in nude mice caused dramatic reduction in tumor size. Taken together, these results implicate nucleolin in the regulation of human astrocytoma proliferation in vitro and tumorigenicity in vivo and suggest that nucleolin may represent a potential novel therapeutic target for astrocytomas.

It's Getting Complicated-A Fresh Look at p53-MDM2-ARF Triangle in Tumorigenesis and Cancer Therapy.

Anti-tumorigenic mechanisms mediated by the tumor suppressor p53, upon oncogenic stresses, are our bodies' greatest weapons to battle against cancer onset and development. Consequently, factors that possess significant p53-regulating activities have been subjects of serious interest from the cancer research community. Among them, MDM2 and ARF are considered the most influential p53 regulators due to their abilities to inhibit and activate p53 functions, respectively. MDM2 inhibits p53 by promoting ubiquitination and proteasome-mediated degradation of p53, while ARF activates p53 by physically interacting with MDM2 to block its access to p53. This conventional understanding of p53-MDM2-ARF functional triangle have guided the direction of p53 research, as well as the development of p53-based therapeutic strategies for the last 30 years. Our increasing knowledge of this triangle during this time, especially through identification of p53-independent functions of MDM2 and ARF, have uncovered many under-appreciated molecular mechanisms connecting these three proteins. Through recognizing both antagonizing and synergizing relationships among them, our consideration for harnessing these relationships to develop effective cancer therapies needs an update accordingly. In this review, we will re-visit the conventional wisdom regarding p53-MDM2-ARF tumor-regulating mechanisms, highlight impactful studies contributing to the modern look of their relationships, and summarize ongoing efforts to target this pathway for effective cancer treatments. A refreshed appreciation of p53-MDM2-ARF network can bring innovative approaches to develop new generations of genetically-informed and clinically-effective cancer therapies.