Abstract Chromosomal instability (CIN) caused by β-Catenin dependent aberrant activation of canonical Wnt signaling or Microsatellite instability (MSI) triggered by inactivation of mismatch repair (MMR) pathway, are the two major genetic instability pathways that drive classical age-related sporadic colorectal cancer (CRC). Activation of canonical Wnt signaling and MSI are primary tumor initiating events in about 80% and 15% of late-onset CRC cases, respectively. Canonical Wnt signaling is also reported as a secondary event in tumors primarily driven by MSI. These inferences are based on seminal studies that included a disproportionately greater number of colon tumors (as compared to rectal tumors). Recent genome-wide studies have suggested colon and rectal cancer to be a single entity, though several other studies appear to indicate otherwise. Exclusive studies on rectal cancer (RC), especially the early-onset sporadic subtype, have been fewer. Despite a recent trend of increased worldwide incidence, early-onset sporadic rectal cancer (EOSRC) is not well understood. We profiled canonical Wnt, KRAS and p53 (components of the classical colorectal carcinoma progression model) and MSI status in a panel of 298 colorectal cancer samples. 41% of EOSRC samples did not harbor Wnt or MSI pathways; the high proportion of a ‘double negative’ entity was neither identified in late-onset RC samples nor in colon cancer samples. KRAS mutation frequency was also significantly lower in EOSRC (24%). Since CIN is a hallmark of canonical Wnt activation driven CRC, we profiled genome-wide DNA copy number alterations (CNAs) in microsatellite stable EOSRC samples and surprisingly identified extensive chromosomal aberrations in both Wnt active and Wnt inactive subtypes suggesting the interesting possibility of presence of CIN in the absence of canonical Wnt activation. Several CNAs were detected exclusively in Wnt inactive samples (being absent in Wnt active samples) and were validated by quantitative PCR. As expected, a few CNAs, such as an amplification detected at 17q12 (ERBB2/GRB7), were present in both subtypes. Genome-wide transcript profiling performed in parallel revealed the elevated expression of genes located within the amplifications, validated by quantitative reverse transcription PCR (Q-RT-PCR). More importantly, aberrant activation of non-canonical signaling pathways was identified in a subset of the ‘double negative’ EOSRC samples, based on unsupervised (hierarchical clustering) and supervised (significance analysis of microarrays and gene set enrichment analysis) analyses of the transcriptome data. Elevation of non-canonical pathway gene transcripts was confirmed by Q-RT-PCR. Our study has therefore revealed presence of unique tumorigenesis pathways in EOSRC samples distinct from canonical pathways that drive late-onset CRC. Citation Format: Ratheesh Raman, Viswakalyan Kotapalli, Raju SR Adduri, Vasantha Kumar Bhaskara, Swarnalata Gowrishankar, Leena Bashyam, Ajay Chaudhary, Mohana Vamsy Chigurupati, Sujith Patnaik, Mukta Srinivasulu, Regulagadda Sastry, Subramanyeshwar Rao, Anjayneyulu Vasala, NarasimhaRaju Kalidindi, Jonathan Pollack, Sudha Murthy, Murali D. Bashyam. Presence of non-canonical tumorigenesis pathways in early-onset sporadic rectal cancer. [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 1198. doi:10.1158/1538-7445.AM2013-1198
Abstract Though primarily a tumor suppressor, TP53 harboring specific missense mutations located in the region encoding the DNA binding domain exhibits a gain of function by transcriptional activation of oncogenes. We performed microarray‐based messenger RNA profiling of squamous cell carcinoma of the oral tongue (SCCOT) and identified significant elevation of SMARCD1 in samples exhibiting p53 nuclear stabilization. Activation of SMARCD1 by mutant p53 was confirmed by evaluation of additional tongue cancer samples as well as The Cancer Genome Atlas expression datasets. SMARCD1 knockdown in HNSCC cells resulted in a significant reduction in several tumorigenic characteristics including cell viability, ability to form colonies in liquid and solid media and cell migration. We identified significantly increased SMARCD1 transcript levels in tumor versus matched normal samples in SCCOT as well as in other cancer types. Increased SMARCD1 expression predicted poor survival in HNSCC tumors harboring missense p53 mutations. Our results suggest SMARCD1 to be a novel transcriptional target of mutant p53.
ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. Further, co-infections with HIV and Mtb have severe effects in the host, with people infected with HIV being fifteen to twenty-one times more likely to develop active TB. The use of an appropriate animal model for HIV/ Mtb co-infection that can recapitulate the diversity of the immune response in humans would be a useful tool for conducting basic and translational research in HIV/ Mtb infections. The present study was focused on developing a humanized mouse model for investigations on HIV- Mtb co-infection. Using NSG-SGM3 mice that can engraft human stem cells, our studies showed that they were able to engraft human CD34+ stem cells which then differentiate into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb , these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced the development of granulomatous lesions in the lungs, detected by CT scan and histopathology. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Our results suggest that the humanized NSG-SGM3 mice are able to recapitulate the effects of HIV and Mtb infections and co-infection in the human host at pathological, immunological and metabolism levels, providing a dependable small animal model for studying HIV/ Mtb co-infection.
Abstract Squamous cell carcinoma of the oral tongue (SCCOT) is a common form of head and neck squamous cell carcinoma (HNSCC). In the last few decades, a steady increase in incidence rates of SCCOT has been reported across the world including India. SCCOT is known to be an age related disease and tobacco consumption is implicated as the main etiological agent, like other HNSCC subtypes. Recent reports have indicated an increase in incidence of SCCOT in the young and in nonsmokers. Among HNSCC, SCCOT appears to be more aggressive with respect to its clinical and biological behavior. Molecular genetic studies on SCCOT are scarce and most studies have been conducted on a small cohort of patients and/or restricted to a single/few molecular marker(s). We have conducted a multipronged molecular genetic study of SCCOT and analyzed the status of known tumorigenesis pathways including TP53, epidermal growth factor receptor (EGFR), microsatellite instability (MSI), CDKN2A, FHIT and human papilloma virus (HPV) infection in surgically resected primary tumor samples and correlated with clinocopathological variables. 67% (67/100) of SCCOT samples exhibited p53 nuclear stabilization whereas a greater proportion (87/100; 87%) exhibited elevated EGFR expression, in accordance with existing literature. Interestingly, p53 nuclear stabilization was found to be more common in young (31/39; 79.5%) than in older (35/59; 59.3%) patients (p = 0.0481). As expected, patients with p53 nuclear stabilization exhibited poorer survival. Further, PCR based mutation screening of exons 5-8 of TP53 (encoding the DNA binding domain) revealed mutations in 50% of samples exhibiting nuclear stabilization. HPV infection was observed in 11/82 (13.4%) tumors while the frequencies of MSI (12/86; 14%) and loss of heterozygosity (LoH) in CDKN2A (32.6%) and FHIT (28.4%) loci were significantly higher than previous studies. In addition, LoH at FHIT locus was significantly associated with p53 nuclear stabilization (p=0.047). Analysis of genome-wide DNA copy number alterations (CNA) using array based comparative genomic hybridization revealed unique copy number alterations validated by quantitative PCR. Exome sequencing and transcriptome profiling are currently underway to understand the pathways/genes deregulated in SCCOT, especially in samples not exhibiting p53 nuclear stabilization. Citation Format: Raju Sr Adduri, Viswakalyan Kotapalli, Neha Ak Gupta, Swarnalata Gowrishankar, Mukta Srinivasulu, Subramanyeshwar Rao, Shantveer G. Uppin, Umanath Karopadi Nayak, Mohana Vamsy Chigurupati, Sujith Chayu Patnaik, NarasimhaRaju Kalidindi, Murali D. Bashyam. Analysis of genetic aberrations in squamous cell carcinoma of the oral tongue. [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 1216. doi:10.1158/1538-7445.AM2013-1216
Two genetic instability pathways viz. chromosomal instability, driven primarily by APC mutation induced deregulated Wnt signaling, and microsatellite instability (MSI) caused by mismatch repair (MMR) inactivation, together account for >90% of late-onset colorectal cancer (CRC). Our understanding of early-onset sporadic CRC is however comparatively limited. In addition, most seminal studies have been performed in the western population and analyses of tumorigenesis pathway(s) causing CRC in developing nations have been rare. We performed a comparative analysis of early and late-onset CRC from India with respect to common genetic aberrations including Wnt, KRAS, and p53 (constituting the classical CRC progression sequence) in addition to MSI. Our results revealed the absence of Wnt and MSI in a significant proportion of early-onset as against late-onset CRC in India. In addition, KRAS mutation frequency was significantly lower in early-onset CRC indicating that a significant proportion of CRC in India may follow tumorigenesis pathways distinct from the classical CRC progression sequence. Our study has therefore revealed the possible existence of non-canonical tumorigenesis pathways in early-onset CRC in India.
Squamous cell carcinoma of tongue (SCCT) is expected to harbor unique clinico-pathological and molecular genetic features since a significant proportion of patients are young and exhibit no association with tobacco or alcohol.We determined P53, epidermal growth factor receptor, microsatellite instability, human papilloma virus infection and loss of heterozygosity status at several tumor suppressor loci in one hundred and twenty one oral SCCT (SSCOT) samples and analyzed their association with clinico-pathological features and patient survival.Our results revealed a significantly higher incidence of p53 nuclear stabilization in early (as against late) onset SCCOT. FHIT loss was significantly associated with p53 nuclear stabilization and the association was stronger in patients with no history of tobacco use. Samples harboring mutation in p53 DNA binding domain or exhibiting p53 nuclear stabilization, were significantly associated with poor survival.Our study has therefore identified distinct features in SCCOT tumorigenesis with respect to age and tobacco exposure and revealed possible prognostic utility of p53.
Chronic kidney disease (CKD) of uncertain etiology (CKDu) is a global health concern affecting tropical farming communities. CKDu is not associated with typical risk factors (e.g., diabetes) and strongly correlates with environmental drivers. To gain potential insights into disease etiology and diagnosis, here we report the first urinary proteome comparing patients with CKDu and non-CKDu controls from Sri Lanka. We found 944 differentially abundant proteins. In silico analyses identified 636 proteins of likely kidney and urogenital origin. As expected, renal tubular injury in patients with CKDu was evinced by increases in albumin, cystatin C, and β2-microglobulin. However, several proteins typically elevated under CKD, including osteopontin and α-N-acetylglucosaminidase, were decreased in patients with CKDu. Furthermore, urinary excretion of aquaporins found higher in CKD was lower in CKDu. Comparisons with previous CKD urinary proteome datasets revealed a unique proteome for CKDu. Notably, the CKDu urinary proteome was relatively similar to that of patients with mitochondrial diseases. Furthermore, we report a decrease in endocytic receptor proteins responsible for protein reabsorption (megalin and cubilin) that correlated with an increase in abundance of 15 of their cognate ligands. Functional pathway analyses identified kidney-specific differentially abundant proteins in patients with CKDu denoted significant changes in the complement cascade and coagulation systems, cell death, lysosomal function, and metabolic pathways. Overall, our findings provide potential early detection markers to diagnose and distinguish CKDu and warrant further analyses on the role of lysosomal, mitochondrial, and protein reabsorption processes and their link to the complement system and lipid metabolism in CKDu onset and progression.NEW & NOTEWORTHY CKDu is a global health concern debilitating a number of tropical rural farming communities. In the absence of typical risk factors like diabetes and hypertension and the lack of molecular markers, it is crucial to identify potential early disease markers. Here, we detail the first urinary proteome profile to distinguish CKDu from CKD. Our data and in silico pathway analyses infer the roles of mitochondrial, lysosomal, and protein reabsorption processes in disease onset and progression.
Purpose The tumor suppressor p53 is known to be inactivated frequently in various cancers. In addition, germline polymorphisms in TP53 are known to affect protein function and influence risk of developing different types of cancers. In this study, we analyzed the association of TP53 Pro72Arg polymorphism with squamous cell carcinoma of oral tongue (SCCOT) and esophagus (ESCC) in India. Methods We assessed the distribution of TP53 Pro72Arg polymorphism in one hundred and fifteen and eighty two SCCOT and ESCC patients, respectively, with respect to one hundred and ten healthy controls from the same population. In addition, we analyzed association of the polymorphism with several clinico-pathological and molecular parameters. Results Pro72 allele was significantly enriched in SCCOT patients compared to the healthy control group but neither allele was enriched in ESCC. Interestingly, Pro72 allele was preferentially mutated in ESCC which was confirmed by analysis of samples heterozygous for Pro72Arg. Conclusions Our study revealed the association of Pro72 allele with SCCOT suggesting the effect of this polymorphism on SCCOT risk. Preferential mutation of Pro72 allele exclusively in ESCC indicates the need for further studies to understand the tissue specific effect of p53 polymorphism.
Abstract Background: Mutations in the TP53 gene are observed at a high frequency in Esophageal Squamous Cell Carcinoma (ESCC) with a majority of alterations being of the missense type affecting residues located in the region encoding the DNA binding domain. These mutations are associated with poor patient survival. However, characterization of the transcriptional networks regulated by p53 mutant proteins is limited to only a few frequent ‘hotspot' mutants, while comparatively rarer mutants have not been characterized. Objectives: Identification and characterization of differentially expressed genes associated with p53 mutation status in ESCC tumor samples. Elucidation of the mechanism of transcriptional regulation of mutant p53 target genes. Methods: A defined set of ESCC tumor samples with known p53 mutation status was selected for this study. The ability of each 'rare' p53 mutation to modulate tumor-related phenotypes was evaluated by performing phenotypic assays such as proliferation, colony formation and migration, in head and neck cancer cell-lines. Gene expression microarray analysis was performed on 36 ESCC tumors to determine putative target genes of 'rare' p53 mutant forms. Following dimension reduction, differentially expressed (putative mutant p53 target) genes were identified using Significance Analysis of Microarrays (SAM) and validated using reverse transcription quantitative PCR (RT-qPCR). Transcription induction of the putative target genes by ectopically expressed wild-type and mutant p53, in p53-null H1299 cell-line, were also evaluated by RT-qPCR. Chromatin immuno-precipitation (ChIP) assay was used to determine localization of mutant p53 proteins to chromosomal loci harboring the putative target genes. Similarly, ability to activate target promoters by mutant p53 was validated through promoter-luciferase assay. Results: Genome-wide analysis revealed 27 differentially expressed genes, of which 9 exhibited up-regulated transcript levels in p53 mutant tumors. Three putative mutant p53 target genes - C1QBP, ARF6 and TRIM23 - were selected for further analysis due to previous reports of their association with cancer. TP53 transcript levels positively correlated with transcript levels of the identified potential mutant targets. The ectopically-expressed p53 mutants - P190T and P278L, induced the increased expression of the target genes, were localized to and activated the promoters of the target genes. Further characterization of the target genes is currently underway. Conclusions: Three novel potential oncogenic targets of mutant p53 proteins were identified. The study further strengthened the heterogeneity in the functioning of different p53 mutants. Citation Format: Sara A. George, Viswakalyan Kotapalli, Raju S. Adduri, Raju Kumar, Sanjana Sarkar, Ramaswamy Pandilla, Murali D. Bashyam. Identification of novel oncogenic targets of mutant p53 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2494.
Surface functionalization of nanoparticles (NPs) may alter their biological interactions such as uptake by alveolar macrophages (AMs).Pulmonary delivery of gold NPs (Au NPs) has theranostic potential due to their optoelectronic properties, minimal alveoli to blood translocation, and possibility of specific cell targeting.Here, we examined whether coating Au NPs with transferrin alters their protein corona, uptake by macrophages, and pulmonary translocation.Methods: Rats were intratracheally instilled with transferrin-coated Au NPs (Tf-Au NPs) or polyethylene glycol-coated Au NPs (PEG-Au NPs).AMs were collected and processed for quantitation of Au cell uptake using ICP-MS and electron microscopy.Au retention in the lungs and other organs was also determined.The uptake of fluorescently labeled Tf-Au NPs and PEG-Au NPs by monocyte-derived human macrophages was also evaluated in vitro.Results: We showed that Tf-Au NPs were endocytosed by AMs and were retained in the lungs to a greater extent than PEG-Au NPs.Both Au NPs acquired similar protein coronas after incubation in rat broncho-alveolar lavage fluid (BALf).The translocation of Au from both NPs to other organs was less than 0.5% of the instilled dose.Transferrin coating enhanced the uptake of Au NPs by primary monocyte-derived human macrophages.Conclusions: We report that coating of NP surface with transferrin can target them to rat AMs and human monocyte-derived macrophages.NP functionalization with transferrin may enhance NP-based therapeutic strategies for lung diseases.
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