Shyam S. Chauhan

Increased Expression and Activity of Nuclear Cathepsin L in Cancer Cells Suggests a Novel Mechanism of Cell Transformation

It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non–cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus. (Mol Cancer Res 2007;5(9):899–907)

iTRAQ-multidimensional liquid chromatography and tandem mass spectrometry-based identification of potential biomarkers of oral epithelial dysplasia and novel networks between inflammation and premalignancy.

Chronic exposure of the oral mucosa to carcinogens in tobacco is linked to inflammation and development of oral premalignant lesions (OPLs) with high risk of progression to cancer; there is currently no clinical methodology to identify high-risk lesions. We hypothesized that identification of differentially expressed proteins in OPLs in relation to normal oral tissues using proteomic approach will reveal changes in multiple cellular pathways and aid in biomarker discovery. Isobaric mass tags (iTRAQ)-labeled oral dysplasias and normal tissues were compared against pooled normal control by online liquid chromatography and tandem mass spectrometry. Verification of biomarkers was carried out in an independent set of samples by immunohistochemistry, immunoblotting, and RT-PCR. We identified 459 nonredundant proteins in OPLs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of three best-performing biomarkers identified by iTRAQ analysis and verified by immunohistochemistrystratifin (SFN), YWHAZ, and hnRNPKachieved a sensitivity of 0.83, 0.91, specificity of 0.74, 0.95, and predictive value of 0.87 and 0.96, respectively, in discriminating dysplasias from normal tissues, thereby confirming their utility as potential OPL biomarkers. Pathway analysis revealed direct interactions between all the three biomarkers and their involvement in two major networks involved in inflammation, signaling, proliferation, regulation of gene expression, and cancer. In conclusion, our work on determining the OPL proteome unraveled novel networks linking inflammation and development of epithelial dysplasia and their key regulatory proteins may serve as novel chemopreventive/therapeutic targets for early intervention. Additionally, we identified and verified a panel of OPL biomarkers that hold promise for large-scale validation for ultimate clinical use.

Heterogeneous ribonucleoprotein K is a marker of oral leukoplakia and correlates with poor prognosis of squamous cell carcinoma

Oral leukoplakia is a heterogeneous lesion with risk of cancer development; there are no biomarkers to predict its potential of malignant transformation. Tissue proteomic analysis of oral leukoplakia using iTRAQ labeling liquid chromatography–mass spectrometry showed overexpression of heterogeneous ribonucleoprotein K (hnRNP K), a transformation‐related RNA‐binding protein, in leukoplakia in comparison with normal tissue. Herein, we investigated the clinical significance of hnRNP K in identification of oral leukoplakic lesions in early stages and as a prognostic marker in head‐and‐neck/oral squamous cell carcinomas (HNOSCCs). Immunohistochemical analysis of hnRNP K was performed in 100 HNOSCCs, 199 leukoplakias and 55 nonmalignant tissues and correlated with clinicopathologic parameters and disease prognosis over 6 years for HNOSCCs. hnRNP K nuclear expression increased from normal tissues to leukoplakia, and frank malignancy (p < 0.001). Cytoplasmic hnRNP K increased significantly from leukoplakia to HNOSCCs (p < 0.001) and was associated with poor prognosis of HNOSCCs (p = 0.011) by Kaplan–Meier analysis. The most important finding of our follow‐up study is that cytoplasmic hnRNP K is an independent predictor of disease recurrence in HNOSCC patients. In conclusion, nuclear hnRNP K may serve as a potential marker for early diagnosis, whereas its cytoplasmic accumulation can help to identify a subgroup of HNOSCC patients with poor prognosis, suggesting its putative utility in clinical management of HNOSCC.

CXCR7 mediated Giα independent activation of ERK and Akt promotes cell survival and chemotaxis in T cells

Chemokine receptors CXCR7 and CXCR4 bind to the same ligand stromal cell derived factor-1alpha (SDF-1α/CXCL12). We assessed the downstream signaling pathways mediated by CXCL12-CXCR7 interaction in Jurkat T cells. All experiments were carried out after functionally blocking the CXCR4 receptor. CXCL12, on binding CXCR7, induced phosphorylation of extra cellular regulated protein kinases (ERK 1/2) and Akt. Selective inhibition of each signal demonstrated that phosphorylated ERK 1/2 is essential for chemotaxis and survival of T cells whereas activation of Akt promotes only cell survival. Another interesting finding of this study is that CXCL12-CXCR7 interaction under normal physiological conditions does not activate the p38 pathway. Furthermore, we observed that the CXCL12 signaling via CXCR7 is Giα independent. Our findings suggest that CXCR7 promotes cell survival and does not induce cell death in T cells. The CXCL12 signaling via CXCR7 may be crucial in determining the fate of the activated T cells.

Nuclear S100A7 Is Associated with Poor Prognosis in Head and Neck Cancer

Background Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC. Methodology Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (ptrend<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3−5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients. Conclusions Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions.

Nuclear and Cytoplasmic Accumulation of Ep-ICD Is Frequently Detected in Human Epithelial Cancers

Background We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. Methodology and Results Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. Conclusions Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers.

Prognostic significance of nuclear pSTAT3 in oral cancer.

Aberrant nuclear accumulation of proteins influences tumor development and may predict biologic aggressiveness and disease prognosis. This study determined the prognostic significance of pSTAT3 (phosphorylayed signal transducer and activator of transcription 3) in oral squamous cell carcinomas (OSCCs).

14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in Head and Neck cancer cells

BackgroundThe five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells.MethodsHead and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies.ResultsUsing in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP).ConclusionGS targets 14-3-3 zeta associated cellular pathways for reducing proliferation and inducing apoptosis in head and neck cancer cells, warranting its investigation for use in treatment of head and neck cancer.

Dipeptidyl peptidase III: a multifaceted oligopeptide N‐end cutter

Dipeptidyl peptidase III (DPP III), the sole member and representative of the M49 family of metallopeptidases, is a zinc‐dependent aminopeptidase. It sequentially hydrolyses dipeptides from the N‐terminal of oligopeptides ranging from three to 10 amino acid residues. Although implicated in an array of pathophysiological phenomena, the precise function of this peptidase is still unclear. However, a number of studies advocate its contribution in terminal stages of protein turnover. Altered expression of DPP III which suggests its involvement in primary ovarian carcinoma, oxidative stress (Nrf2 nuclear localization), pain, inflammation and cataractogenesis has recently led to resurgence of interest in delineating the role of the peptidase in these pathophysiological processes. This review article intends to bring forth the latest updates in this arena which may serve as a base for future studies on the peptidase.

High Levels of Cell-Free Circulating Nucleic Acids in Pancreatic Cancer are Associated With Vascular Encasement, Metastasis and Poor Survival

Pancreatic cancer is a highly aggressive disease with rapid invasion and early encasement of blood vessels. Hence, levels of circulating nucleic acids and tumor-associated mutations in them may have clinical importance. We analyzed the levels of circulating tumor DNA and oncogenic k-ras mutation in plasma of patients with pancreatic cancer and correlated their levels with survival and clinicopathological parameters. Higher levels of plasma DNA (>62 ng/mL) was found to associate significantly with lower overall survival time (p = .002), presence of vascular encasement (p = .030) and metastasis (p = .001). However, k-ras mutation status did not correlate with any of the clinicopathological parameters or survival. We conclude that circulating DNA in plasma can be an important predictor of prognosis in pancreatic cancer.