Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The spatial and temporal distributions of proteins are critical to protein function, but cannot be directly assessed by measuring protein bundance. Here we describe a mass spectrometry-based proteomics strategy, Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently protein turnover rates and subcellular localization in the same experiment. Applying the method, we find that unfolded protein response (UPR) has different effects on protein turnover dependent on their subcellular location in human AC16 cells, with proteome-wide slowdown but acceleration among stress response proteins in the ER and Golgi. In parallel, UPR triggers broad differential localization of proteins including RNA-binding proteins and amino acid transporters. Moreover, we observe newly synthesized proteins including EGFR that show a differential localization under stress than the existing protein pools, reminiscent of protein trafficking disruptions. We next applied SPLAT to an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model of cancer drug cardiotoxicity upon treatment with the proteasome inhibitor carfilzomib. Paradoxically, carfilzomib has little effect on global average protein half-life, but may instead selectively disrupt sarcomere protein homeostasis. This study provides a view into the interactions of protein spatial and temporal dynamics and demonstrates a method to examine protein homeostasis regulations in stress and drug response.

Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins. A global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, termed Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge in human AC16 cells, we find that the changes in protein turnover kinetics during UPR varies across subcellular localizations, with overall slowdown but an acceleration in endoplasmic reticulum and Golgi proteins involved in stress response. In parallel, the spatial proteomics component of the experiment revealed an externalization of amino acid transporters and ion channels under UPR, as well as the migration of RNA-binding proteins toward an endosome co-sedimenting compartment. The SPLAT experimental design classifies heavy and light SILAC labeled proteins separately, allowing the observation of differential localization of new and old protein pools and capturing a partition of newly synthesized EGFR and ITGAV to the ER under stress that suggests protein trafficking disruptions. Finally, application of SPLAT toward human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) exposed to the cancer drug carfilzomib, identified a selective disruption of proteostasis in sarcomeric proteins as a potential mechanism of carfilzomib-mediated cardiotoxicity. Taken together, this study provides a global view into the spatiotemporal dynamics of human cardiac cells and demonstrates a method for inferring the coordinations between spatial and temporal proteome regulations in stress and drug response.

Critical pathways of oral squamous cell carcinoma: molecular biomarker and therapeutic intervention.

The rapid growth of oral cancer is a significant concern, especially in developing countries due to the advanced lifestyle and 5-year survival despite advanced multimodality of cancer care. The poor modality might be due to the detection of disease in the advanced stage. Early detection and development of novel therapies can improve oral cancer patient survival. The PI3K/AKT/mTOR and RAS-RAF-MEK-ERK are very extensively exploited pathways in oral cancer. These pathways are very critical in the progression of tumorigenesis in oral cancer. This review focuses on the association of Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways in terms of protein expression level, genetic mutation, and therapeutic intervention in oral cancer.

Lung cancer in the older population:Interactive effects of angiotensin converting enzyme gene polymorphism (rs 4340 ID) and tobacco addiction in risk assessment.

BACKGROUND: rs4340ID polymorphism of angiotensin-converting enzyme (ACE) correlates with serum ACE levels in many known cancers. This study analyzed ACE rs4340 ID polymorphism in lung cancer (LC) in older patients of North India and correlated it with addiction status. METHODS: The study enrolled all subjects aged 60 years and above with 154 LC and 205 healthy controls. Genotyping was done by polymerase chain reaction (PCR) and validated by sequencing of 10% of the sample. Statistical analysis was done by SPSS Statistics 21. RESULTS: Genotype II was observed to have a significant 2.21-fold increased risk of LC as compared to the DD genotype and 3.43-folds enhanced risk with interaction of I allele with tobacco consumption habits as compared to D allele in LC was seen. CONCLUSION: The risk of LC was higher with II genotype as compared to DD genotype. Interactive effect showed that I allele with tobacco habits may increase the risk of LC.

Serum sHLA-G: Significant diagnostic biomarker with respect to therapy and immunosuppressive mediators in Head and Neck Squamous Cell Carcinoma.

Head & Neck Squamous Cell Carcinoma is one of the highest mortality factors in the world due to the lack of potential biomarker for early detection of disease. There is an urgent need for molecular marker involved in disease progression which remains suppressed normally, required for specificity. HLA-G is highly expressed in cancers and creates immune-suppressive microenvironment. Cancerous cells secrete inflammatory cytokines like IL-10,IFN-γ which increase expression of immunosuppressive molecules, such as HLA-G. We evaluated sHLA-G protein level in serum of 120 HNSCC patients at diagnosis and after therapy and compared with 99 individuals by SPR, ELISA and determined its mRNA level by qRT-PCR. sHLA-G was correlated with serum IL-10 and IFN-γ of the patients. Significant elevated levels of sHLA-G were observed in patients (8.25 ± 1.74 ng/µl) than control (6.45 ± 1.31 ng/µl). Levels were declined in (8.09 ± 1.79 ng/µl to 6.64 ± 1.33 ng/µl) patients in response to therapy. sHLA-G levels with tumor burden (8.16 ± 1.91 to 6.63 ± 1.32 ng/µl), node (8.62 ± 1.45 to 6.66 ± 1.26 ng/µl), PDSCC (8.14 ± 0.62 to 5.65 ± 0.27 ng/µl) and oropharynx (7.90 ± 1.24 to 6.10 ± 1.33 ng/µl) showed a positive and significant response to therapy. Findings indicate that sHLA-G can be a potential diagnostic serum protein marker for HNSCC due to its suppressive function and over expression in diseased condition with the influence of cytokines.

Diagnostic Significance of p38 Isoforms (p38α, p38ß, p38γ, p38δ) in Head and Neck Squamous Cell Carcinoma: Comparative Serum Level Evaluation and Design of Novel Peptide Inhibitor Targeting the Same.

PURPOSE: The p38 mitogen-activated protein kinase (MAPKs) play a crucial role in the production of pro-inflammatory cytokines and over-expression of it increase cytokines which promote cancer. Among four isoforms, p38α has been well studied in head and neck squamous cell carcinoma (HNSCC) and other cancers as a therapeutic target. p38δ has recently emerged as a potential disease-specific drug target. Elevated serum p38α level in HNSCC was reported earlier from our lab. This study aims to estimate the levels of p38 MAPK-isoforms in the serum of HNSCC and design peptide inhibitor targeting the same. MATERIALS AND METHODS: Levels of p38 MAPK isoforms in the serum of HNSCC and healthy controls were quantified by surface plasmon resonance technology. The peptide inhibitor for p38 MAPK was designed by molecular modeling using Grid-based Ligand Docking with Energetics tools and compared with known specific inhibitors. RESULTS: We have observed highly elevated levels of all four isoforms of p38 MAPK in serum of HNSCC patients compared to the control group. Further, serum p38α, p38ß, and p38δ levels were down regulated after therapy in follow-up patients, while p38γ showed no response to the therapy. Present study screened designed peptide WFYH as a specific inhibitor against p38δ. The specific inhibitor of p38δ was found to have no effect on p38α due to great structural difference at ATP binding pocket. CONCLUSION: In this study, first time estimated the levels of p38 MAPK isoforms in the serum of HNSCC. It can be concluded that p38 MAPK isoforms can be a diagnostic and prognostic marker for HNSCC and p38δ as a therapeutic target.

Effects of Tobacco Habits on the Polymorphism of NFKB1 and NFKB1A Gene of Head and Neck Squamous Cell Carcinoma in Indian Population

Background: Polymorphism of NFKB1 and NFKB1A are highly associated with cancer. We have assessed polymorphism in the promoter region of NFKB1 -94 del/ins ATTG (rs28362491) and NFKB1A -826 C/T (rs2233406) with the risk of HNSCC in Indian population. Methods: Polymerase chain reaction­restriction fragment length polymorphism (PCR-RFLP) method was used for the genotyping NFKB1 -94 del/ins ATTG and NFKB1A -826 C/T. Sequencing was done to validate the results of PCR-RFLP. Statistical analysis of data was done by Stata/SE-14.0 software. Results: ins/ins genotype was observed to be a risk factor of HNSCC as compared del/del genotype of NFKB1 -94 ATTG. Interactive effects of smoking and chewing on ins/ins genotype showed 13.96 and 10.92 fold increased risk of HNSCC. NFKB1A -826 C/T polymorphism, TT genotype showed no association with the risk of HNSCC as compared to wild type CC genotype. Conclusion: Our results showed NFKB1 -94 del/ins ATTG with smoking and tobacco chewing may increase the risk of HNSCC while NFKB1A -826 C/T plays a protective role in Indian population.

Promising link of HLA-G polymorphism, tobacco consumption and risk of Head and Neck Squamous Cell Carcinoma (HNSCC) in North Indian population.

Human leukocyte antigen (HLA-G) is a potent immune-tolerant molecule and has a critical role in various pathological conditions of cancer. The aim of the study was to analyze the association of HLA-G polymorphism as a risk factor in Head and Neck Squamous Cell Carcinoma (HNSCC). The HLA-G polymorphism at 3'UTR 14bp INDEL (rs371194629) and +3142G/C (rs1063320) were studied in 383 HNSCC patients and 383 ethnically similar-aged healthy controls in North Indian population. The genotyping study of two polymorphisms of HLA-G was documented using DNA-PAGE and RFLP-PCR method. 14bp INDEL Del/Ins, Ins/Ins genotype and Ins allele were more pronounced in HNSCC patients in compared to controls. Whereas, +3142 C/C genotype and C allele were associated with risk factors in HNSCC. Furthermore, the dual effect of polymorphisms; both variants (Del/Ins-Ins/Ins & G/C-C/C) carrying loci was significantly (OR=2.78) associated with the disease compared to one variant (Del/Del-G/C or Del/Del-C/C or Ins/Ins-G/G). Moreover, both polymorphisms showed promising link in terms of tobacco influence on HNSCC risk. It can be concluded that this study first time reports that C/C, Del/Ins and Ins/Ins genotype as well as C and Ins allele could be major risk factors with strong impact of tobacco for HNSCC in North Indian population.

NFκB-p50 as a blood based protein marker for early diagnosis and prognosis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the major health concern in Indian population. Despite of advanced treatment the mortality rate for this disease has not been improved very much. Current research focused on development of protein marker for the diagnosis and prognosis of HNSCC. The case control study was performed with 125 HNSCC patients and 104 control cases. The level of p50 and IκBα proteins in serum were evaluated at pre and post therapy by label free real time surface plasmon resonance (SPR) and western blot analysis. The serum p50 concentration were significantly (P < 0.0001) higher at the time of diagnosis i.e. pre therapy (Mean ± SD = 27.06 ± 4.88 ng/µl) as compared to controls (Mean ± SD = 16.96 ± 4.04 ng/µl) while it decline at post therapy (Mean ± SD = 21.01 ± 4.98 ng/µl). Similarly, the concentration of IκBα protein in serum were slightly higher at pre therapy (Mean ± SD = 8.33 ± 1.85 ng/µl) as compared to controls (Mean ± SD = 7.27 ± 1.84 ng/µl) and declined at post therapy (Mean ± SD = 7.09 ± 1.24 ng/µl). The level of p50 was also high at the early stage of the disease. The specificity and sensitivity of p50 proteins obtained from ROC analysis revealed the potentiality to be diagnostic protein marker for HNSCC for its accuracy in the study cohort.