Juhi Ojha

Sequestration of Toxic Oligomers by HspB1 as a Cytoprotective Mechanism

ABSTRACT Small heat shock proteins (sHsps) are molecular chaperones that protect cells from cytotoxic effects of protein misfolding and aggregation. HspB1, an sHsp commonly associated with senile plaques in Alzheimers disease (AD), prevents the toxic effects of Aβ aggregates in vitro. However, the mechanism of this chaperone activity is poorly understood. Here, we observed that in two distinct transgenic mouse models of AD, mouse HspB1 (Hsp25) localized to the penumbral areas of plaques. We have demonstrated that substoichiometric amounts of human HspB1 (Hsp27) abolish the toxicity of Aβ oligomers on N2a (mouse neuroblastoma) cells. Using biochemical methods, spectroscopy, light scattering, and microscopy methods, we found that HspB1 sequesters toxic Aβ oligomers and converts them into large nontoxic aggregates. HspB1 was overexpressed in N2a cells in response to treatment with Aβ oligomers. Cultured neurons from HspB1-deficient mice were more sensitive to oligomer-mediated toxicity than were those from wild-type mice. Our results suggest that sequestration of oligomers by HspB1 constitutes a novel cytoprotective mechanism of proteostasis. Whether chaperone-mediated cytoprotective sequestration of toxic aggregates may bear clues to plaque deposition and may have potential therapeutic implications must be investigated in the future.

Behavioral Defects in Chaperone-Deficient Alzheimer's Disease Model Mice

Molecular chaperones protect cells from the deleterious effects of protein misfolding and aggregation. Neurotoxicity of amyloid-beta (Aβ) aggregates and their deposition in senile plaques are hallmarks of Alzheimers disease (AD). We observed that the overall content of αB-crystallin, a small heat shock protein molecular chaperone, decreased in AD model mice in an age-dependent manner. We hypothesized that αB-crystallin protects cells against Aβ toxicity. To test this, we crossed αB-crystallin/HspB2 deficient (CRYAB-/-HSPB2-/-) mice with AD model transgenic mice expressing mutant human amyloid precursor protein. Transgenic and non-transgenic mice in chaperone-sufficient or deficient backgrounds were examined for representative behavioral paradigms for locomotion and memory network functions: (i) spatial orientation and locomotion was monitored by open field test; (ii) sequential organization and associative learning was monitored by fear conditioning; and (iii) evoked behavioral response was tested by hot plate method. Interestingly, αB-crystallin/HspB2 deficient transgenic mice were severely impaired in locomotion compared to each genetic model separately. Our results highlight a synergistic effect of combining chaperone deficiency in a transgenic mouse model for AD underscoring an important role for chaperones in protein misfolding diseases.

Genetic variation associated with longer telomere length increases risk of chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Shorter mean telomere length in leukemic cells has been associated with more aggressive disease. Germline polymorphisms in telomere maintenance genes affect telomere length and may contribute to CLL susceptibility. Methods: We collected genome-wide data from two groups of patients with CLL (N = 273) and two control populations (N = 5,725). In ancestry-adjusted case–control comparisons, we analyzed eight SNPs in genes definitively associated with inter-individual variation in leukocyte telomere length (LTL) in prior genome-wide association studies: ACYP2, TERC, NAF1, TERT, OBFC1, CTC1, ZNF208, and RTEL1. Results: Three of the eight LTL-associated SNPs were associated with CLL risk at P < 0.05, including those near: TERC [OR, 1.46; 95% confidence interval (CI), 1.15–1.86; P = 1.8 × 10−3], TERT (OR = 1.23; 95% CI, 1.02–1.48; P = 0.030), and OBFC1 (OR, 1.36; 95% CI, 1.08–1.71; P = 9.6 × 10−3). Using a weighted linear combination of the eight LTL-associated SNPs, we observed that CLL patients were predisposed to longer LTL than controls in both case–control sets (P = 9.4 × 10−4 and 0.032, respectively). CLL risk increased monotonically with increasing quintiles of the weighted linear combination. Conclusions: Genetic variants in TERC, TERT, and OBFC1 are associated with both longer LTL and increased CLL risk. Because the human CST complex competes with shelterin for telomeric DNA, future work should explore the role of OBFC1 and other CST complex genes in leukemogenesis. Impact: A genetic predisposition to longer telomere length is associated with an increased risk of CLL, suggesting that the role of telomere length in CLL etiology may be distinct from its role in disease progression. Cancer Epidemiol Biomarkers Prev; 25(7); 1043–9. ©2016 AACR.

GWAS in childhood acute lymphoblastic leukemia reveals novel genetic associations at chromosomes 17q12 and 8q24.21

Childhood acute lymphoblastic leukemia (ALL) (age 0–14 years) is 20% more common in Latino Americans than non-Latino whites. We conduct a genome-wide association study in a large sample of 3263 Californian children with ALL (including 1949 of Latino heritage) and 3506 controls matched on month and year of birth, sex, and ethnicity, and an additional 12,471 controls from the Kaiser Resource for Genetic Epidemiology Research on Aging Cohort. Replication of the strongest genetic associations is performed in two independent datasets from the Children’s Oncology Group and the California Childhood Leukemia Study. Here we identify new risk loci on 17q12 near IKZF3/ZPBP2/GSDMB/ORMDL3, a locus encompassing a transcription factor important for lymphocyte development (IKZF3), and at an 8q24 region known for structural contacts with the MYC oncogene. These new risk loci may impact gene expression via local (four 17q12 genes) or long-range (8q24) interactions, affecting function of well-characterized hematopoietic and growth-regulation pathways.Childhood acute lymphoblastic leukemia is common in Latino Americans. Here, the authors conduct a genome-wide association study in a Californian cohort containing children of Latino heritage, and identify loci on 17q12 and 8q24 which may affect hematopoietic and growth-regulation pathways.

Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8×10−4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches.

TIMP-1 downregulation modulates miR-125a-5p expression and triggers the apoptotic pathway

Matrix metalloproteinases and their natural inhibitors (TIMPs) are important elements in a wide range of oncology settings. Elevated levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) have often been associated with increased tumorigenesis. This has been demonstrated in a number of clinical and experimental models which include breast, gastric, colorectal and non-small cell lung carcinoma (NSCLC). Our earlier studies have identified increased angiogenic activity and aggressive tumor kinetics in TIMP-1 overexpressing H2009 lung adenocarcinoma cells. TIMP-1 overexpression has also been implicated in antiapoptotic responses, inducing a significant upregulation of Bcl-2. These TIMP-1 functions have been shown to be MMP-independent and provide insight into its pleiotropic activities. The current study examines microRNA (miRNA) interactions with this molecule. We have sought to define the relationship between TIMP-1 and miRNA by knocking down TIMP-1 in high TIMP-1 expressing lung adenocarcinoma cell lines. TIMP-1 knockdown resulted in increased expression of miR-125a-5p with a concomitant increase in apoptosis and attenuation of the tumorigenic features of these cells. We have identified TIMP-1 as a bona fide target of miR-125a-5p, and their interaction resulted in an increase in p53 expression. We further corroborated our in vitro data with patient samples, which exhibited an inverse correlation between TIMP-1 and miR-125a-5p expression. Our study lends support to the notion that elevated TIMP-1 levels, which are frequently associated with poor prognosis, cause aberrant modulation of miRNAs.

Abstract 3086: Modulation of microRNA profile in lung adenocarcinoma cell line following TIMP-1 downregulation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Non-small cell carcinoma accounts for 85% of lung cancer cases and has a 5 year survival rate of only 17%. In recent years, tissue inhibitor of metalloproteinase-1 (TIMP-1) has emerged as an important prognostic marker. In several cancers including non-small cell lung cancer, high serum levels of TIMP-1 have been associated with poor prognosis. Previously, we have shown that overexpressing TIMP-1 in H2009, a lung adenocarcinoma cell line resulted in aggressive tumors when implanted in the mouse brain. In the present study, we have knocked down TIMP-1 expression in the non-small cell lung cancer cell line A549 using shRNA. In vitro assays using the TIMP-1 knockdown clones defined decreased migration in wound assays (p<0.01), as well as reduced invasion (p<0.05) using Boyden chamber assays. We investigated these functional alterations by examining microRNAs in these cell lines. MicroRNAs represent a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level and control crucial signaling and physiological processes functioning either as oncogenes or tumor-suppressors. The miRNA expression profile of A549 cell line and its TIMP1 knockdown clone were evaluated using the Affymetrix miRNA microarray platform on Gene-Chip miRNA 3.0 array in A549 cell line. Following hybridization and data acquisition, we used Partek Genomics Suite ® software for RNA normalization and to determine statistically significant differences in miRNA expression between experimental groups by ANOVA and pairwise comparisons (two-sidedα=0.05). Using the Affymetrix miRNA array we have identified altered miRNAs expression in the clones, with 72 differentially regulated miRNAs following TIMP-1 knockdown. Using quantitative real time PCR we have selectively validated several of the miRNAs that showed the maximal difference. We have confirmed a 2 to 4-fold down regulation of MiR-10b, miR-29a and miR-30 and a 3-fold upregulation of miR-125a-5p. Based on KEGG pathway analysis, this group of validated miRNAs are involved in the MAPK signaling pathway. Western blot analysis showed activation of the apoptosis inducing p38 MAPK pathway in the knockdown clones. Additional mechanistic dissection of these changes and involved pathways will contribute further to our understanding of the role of TIMP-1 in lung cancer. (This research was supported by a Georgia Distinguished Cancer Scholar award to AMR). Citation Format: Sampa Ghoshal-Gupta, Ammar Kutiyanawalla, Shahanawaz Jiwani, Juhi Ojha, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. Modulation of microRNA profile in lung adenocarcinoma cell line following TIMP-1 downregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3086. doi:10.1158/1538-7445.AM2013-3086