Activated Epidermal Growth Factor Receptor as a Novel Target in Pancreatic Cancer Therapy

Pancreatic cancer is one of the most fatal among all solid malignancies. Targeted therapeutic approaches have the potential to transform cancer therapy as exemplified by the success of several tyrosine kinase inhibitors. Prompted by this, comprehensive profiling of tyrosine kinases and their substrates was carried out using a panel of low passage pancreatic cancer cell lines. One of the pancreatic cancer cell lines, P196, which showed dramatic upregulation of tyrosine kinase activity as compared to non-neoplastic cells, was systematically studied using a quantitative proteomic approach called stable isotope labeling with amino acids in cell culture (SILAC). A careful analysis of activated tyrosine kinase pathways revealed aberrant activation of epidermal growth factor receptor pathway in this cell line. Mouse xenograft based studies using EGFR inhibitor erlotinib confirmed EGFR pathway to be responsible for proliferation in these tumors. By a systematic study across low passage pancreatic cancer cell lines and mice carrying pancreatic cancer xenografts, we have demonstrated activated epidermal growth factor receptor as an attractive candidate for targeted therapy in a subset of pancreatic cancers. Further, we propose immunohistochemical labeling of activated EGFR (pEGFR¹⁰⁶⁸) as an efficient screening tool to select patients who are more likely to respond to EGFR inhibitors

Proteins with Altered Levels in Plasma from Glioblastoma Patients as Revealed by iTRAQ-Based Quantitative Proteomic Analysis

<div><p>Glioblastomas (GBMs) are the most common and lethal primary tumors of the central nervous system with high level of recurrence despite aggressive therapy. Tumor-associated proteins/peptides may appear in the plasma of these patients as a result of disruption of the blood-brain barrier in them, raising the scope for development of plasma-based tests for diagnosis and monitoring the disease. With this objective, we analyzed the levels of proteins present in the plasma from GBM patients using an iTRAQ based LC-MS/MS approach. Analysis with pooled plasma specimens from the patient and healthy control samples revealed high confidence identification of 296 proteins, of which 61 exhibited a fold-change ≥1.5 in the patient group. Forty-eight of them contained signal sequence. A majority have been reported in the differentially expressed transcript or protein profile of GBM tissues; 6 have been previously studied as plasma biomarkers for GBM and 16 for other types of cancers. Altered levels of three representative proteins–ferritin light chain (FTL), S100A9, and carnosinase 1 (CNDP1)–were verified by ELISA in a test set of ten individual plasma specimens. FTL is an inflammation marker also implicated in cancer, S100A9 is an important member of the Ca²⁺ signaling cascade reported to be altered in GBM tissue, and CNDP1 has been reported for its role in the regulation of the levels of carnosine, implicated as a potential drug for GBM. These and other proteins in the dataset may form useful starting points for further clinical investigations for the development of plasma-based biomarker panels for GBM.</p> </div

Heterogeneous Nuclear Ribonucleoproteins and Their Interactors Are a Major Class of Deregulated Proteins in Anaplastic Astrocytoma: A Grade III Malignant Glioma

Anaplastic astrocytoma is a high grade malignant glioma (WHO grade III) of the central nervous system which arises from a low grade II tumor and invariably progresses into lethal glioblastoma (WHO grade IV). We have studied differentially expressed proteins from the microsomal fraction of the clinical specimens of these tumors, using iTRAQ and high-resolution mass spectrometry followed by immunohistochemistry for representative proteins on tissue sections. A total of 2642 proteins were identified, 266 of them with minimum 2 peptide signatures and 2-fold change in expression. The major groups of proteins revealed to be differentially expressed were associated with key cellular processes such as post transcriptional processing, protein translation, and acute phase response signaling. A distinct inclusion among these important proteins is 10 heterogeneous nuclear ribonucleoproteins (hnRNPs) and their interacting partners which have regulatory functions in the cell. hnRNP-mediated post transcriptional events are known to play a major role in mRNA processing, stability, and distribution. Their altered levels have also been observed by us in lower (diffused astrocytoma) and higher (glioblastoma) grades of gliomas, and membrane localization of hnRNPs has also been documented in the literature. hnRNPs may thus be major factors underlying global gene expression changes observed in glial tumors while their differential presence in the microsomal fraction suggests yet additional and unknown roles in tumorigenesis

Log₂ transformed fold changes for the proteins observed with differential levels in the plasma from GBM patients.

<p>Panel A represents up regulated proteins and Panel B down regulated proteins.</p

Scatter plot representing altered levels of ferritin light chain, S100A9 and carnosinase 1, in individual specimens from control subjects and GBM patients as determined by ELISA.

<p>Elevated levels of Ferritin light chain were observed in 7 out of 10 GBM cases and in 8 out of 10 patients for S100A9. Lower levels of Carnosinase 1were observed in 8 out of 10 GBM patients. The fold changes are shown in log₂ transformed ratio. The details of ELISA are given under Methods.</p

Heterogeneous Nuclear Ribonucleoproteins and Their Interactors Are a Major Class of Deregulated Proteins in Anaplastic Astrocytoma: A Grade III Malignant Glioma

Anaplastic astrocytoma is a high grade malignant glioma (WHO grade III) of the central nervous system which arises from a low grade II tumor and invariably progresses into lethal glioblastoma (WHO grade IV). We have studied differentially expressed proteins from the microsomal fraction of the clinical specimens of these tumors, using iTRAQ and high-resolution mass spectrometry followed by immunohistochemistry for representative proteins on tissue sections. A total of 2642 proteins were identified, 266 of them with minimum 2 peptide signatures and 2-fold change in expression. The major groups of proteins revealed to be differentially expressed were associated with key cellular processes such as post transcriptional processing, protein translation, and acute phase response signaling. A distinct inclusion among these important proteins is 10 heterogeneous nuclear ribonucleoproteins (hnRNPs) and their interacting partners which have regulatory functions in the cell. hnRNP-mediated post transcriptional events are known to play a major role in mRNA processing, stability, and distribution. Their altered levels have also been observed by us in lower (diffused astrocytoma) and higher (glioblastoma) grades of gliomas, and membrane localization of hnRNPs has also been documented in the literature. hnRNPs may thus be major factors underlying global gene expression changes observed in glial tumors while their differential presence in the microsomal fraction suggests yet additional and unknown roles in tumorigenesis

Heterogeneous Nuclear Ribonucleoproteins and Their Interactors Are a Major Class of Deregulated Proteins in Anaplastic Astrocytoma: A Grade III Malignant Glioma

Anaplastic astrocytoma is a high grade malignant glioma (WHO grade III) of the central nervous system which arises from a low grade II tumor and invariably progresses into lethal glioblastoma (WHO grade IV). We have studied differentially expressed proteins from the microsomal fraction of the clinical specimens of these tumors, using iTRAQ and high-resolution mass spectrometry followed by immunohistochemistry for representative proteins on tissue sections. A total of 2642 proteins were identified, 266 of them with minimum 2 peptide signatures and 2-fold change in expression. The major groups of proteins revealed to be differentially expressed were associated with key cellular processes such as post transcriptional processing, protein translation, and acute phase response signaling. A distinct inclusion among these important proteins is 10 heterogeneous nuclear ribonucleoproteins (hnRNPs) and their interacting partners which have regulatory functions in the cell. hnRNP-mediated post transcriptional events are known to play a major role in mRNA processing, stability, and distribution. Their altered levels have also been observed by us in lower (diffused astrocytoma) and higher (glioblastoma) grades of gliomas, and membrane localization of hnRNPs has also been documented in the literature. hnRNPs may thus be major factors underlying global gene expression changes observed in glial tumors while their differential presence in the microsomal fraction suggests yet additional and unknown roles in tumorigenesis

Workflow used to study differential levels of proteins in plasma from GBM patients using iTRAQ technology.

<p>Workflow used to study differential levels of proteins in plasma from GBM patients using iTRAQ technology.</p

MS/MS spectra of select peptides with their reporter ions for three proteins - ferritin light chain, S100A9 and carnosinase 1.

<p>In the analysis, iTRAQ reporter ions114 and 115 represent control specimens whereas 116 and 117 represent plasma from GBM patients.</p

Proteogenomic Analysis of <i>Candida glabrata</i> using High Resolution Mass Spectrometry

<i>Candida glabrata</i> is a common opportunistic human pathogen leading to significant mortality in immunosuppressed and immunodeficient individuals. We carried out proteomic analysis of <i>C. glabrata</i> using high resolution Fourier transform mass spectrometry with MS resolution of 60000 and MS/MS resolution of 7500. On the basis of 32453 unique peptides identified from 118815 peptide–spectrum matches, we validated 4421 of the 5283 predicted protein-coding genes (83%) in the <i>C. glabrata</i> genome. Further, searching the tandem mass spectra against a six frame translated genome database of <i>C. glabrata</i> resulted in identification of 11 novel protein coding genes and correction of gene boundaries for 14 predicted gene models. A subset of novel protein-coding genes and corrected gene models were validated at the transcript level by RT-PCR and sequencing. Our study illustrates how proteogenomic analysis enabled by high resolution mass spectrometry can enrich genome annotation and should be an integral part of ongoing genome sequencing and annotation efforts