Establishment of a diverse head and neck squamous cancer cell bank using conditional reprogramming culture methods.

Most laboratory models of head and neck squamous cell cancer (HNSCC) rely on established immortalized cell lines, which carry inherent bias due to selection and clonality. We established a robust panel of HNSCC tumor cultures using a "conditional reprogramming" (CR) method, which utilizes a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Sixteen CR cultures were successfully generated from 19 consecutively enrolled ethnically and racially diverse patients with HNSCC at a tertiary care center in the Bronx, NY. Of the 16 CR cultures, 9/16 were derived from the oral cavity, 4/16 were derived from the oropharynx, and 3/16 were from laryngeal carcinomas. Short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue DNA. All CR cultures expressed ΔNp63 and cytokeratin 5/6, which are markers of squamous identity. Human papillomavirus (HPV) testing was assessed utilizing clinical p16 staining on primary tumors, reverse transcription polymerase chain reaction (RT-PCR) of HPV16/18-specific viral oncogenes E6 and E7 in RNA extracted from tumor samples, and HPV DNA sequencing. Three of four oropharyngeal tumors were p16 and HPV-positive and maintained HPV in culture. CR cultures were able to establish three-dimensional spheroid and murine flank and orthotopic tongue models. CR methods can be readily applied to all HNSCC tumors regardless of patient characteristics, disease site, and molecular background, providing a translational research model that properly includes patient and tumor diversity.

miR-375 Regulates Invasion-Related Proteins Vimentin and L-Plastin.

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our in vitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion in vitro, indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.

Combined P16 and human papillomavirus testing predicts head and neck cancer survival.

While its prognostic significance remains unclear, p16(INK4a) protein expression is increasingly being used as a surrogate marker for oncogenic human papillomavirus (HPV) infection in head and neck squamous cell carcinomas (HNSCC). To evaluate the prognostic utility of p16 expression in HNSCC, we prospectively collected 163 primary tumor specimens from histologically confirmed HNSCC patients who were followed for up to 9.4 years. Formalin fixed tumor specimens were tested for p16 protein expression by immunohistochemistry (IHC). HPV type-16 DNA and RNA was detected by MY09/11-PCR and E6/E7 RT-PCR on matched frozen tissue, respectively. P16 protein expression was detected more often in oropharyngeal tumors (53%) as compared with laryngeal (24%), hypopharyngeal (8%) or oral cavity tumors (4%; p<0.0001). With respect to prognosis, p16-positive oropharyngeal tumors exhibited significantly better overall survival than p16-negative tumors (log-rank test p=0.04), whereas no survival benefit was observed for nonoropharyngeal tumors. However, when both p16 and HPV DNA test results were considered, concordantly positive nonoropharyngeal tumors had significantly better disease-specific survival than concordantly negative nonoropharyngeal tumors after controlling for sex, nodal stage, tumor size, tumor subsite, primary tumor site number, smoking and drinking [adjusted hazard ratio (HR)=0.04, 0.01-0.54]. Compared with concordantly negative nonoropharyngeal HNSCC, p16(+)/HPV16(-) nonoropharyngeal HNSCC (n=13, 7%) demonstrated no significant improvement in disease-specific survival when HPV16 was detected by RNA (adjusted HR=0.83, 0.22-3.17). Our findings show that p16 IHC alone has potential as a prognostic test for oropharyngeal cancer survival, but combined p16/HPV testing is necessary to identify HPV-associated nonoropharyngeal HNSCC with better prognosis.

Characterization of a Diverse Set of Conditionally Reprogrammed Head and Neck Cancer Cell Cultures.

OBJECTIVE: To establish and characterize a diverse library of head and neck squamous cell cancer (HNSCC) cultures using conditional reprogramming (CR). METHODS: Patients enrolled on an IRB-approved protocol to generate tumor cell cultures using CR methods. Tumor and blood samples were collected and clinical information was recorded. Successful CR cultures were validated against banked reference tumors with short tandem repeat genotyping. Cell morphology was archived with photodocumentation. Clinical and demographic factors were evaluated for associations with successful establishment of CR culture. Human papilloma virus (HPV) genotyping, clonogenic survival, MTT assays, spheroid growth, and whole exome sequencing were carried out in selected cultures. RESULTS: Forty four patients were enrolled, with 31 (70%) successful CR cultures, 32% derived from patients who identified as Black and 61% as Hispanic. All major head and neck disease sites were represented, including 15 (48%) oral cavity and 8 (26%) p16-positive oropharynx cancers. Hispanic ethnicity and first primary tumors (vs. second primary or recurrent tumors) were significantly associated with successful CR culture. HPV expression was conserved in CR cultures, including CR-024, which carried a novel HPV-69 serotype. CR cultures were used to test cisplatin responses using MTT assays. Previous work has also demonstrated these models can be used to assess response to radiation and can be engrafted in mouse models. Whole exome sequencing demonstrated that CR cultures preserved tumor mutation burden and driver mutations. CONCLUSION: CR culture is highly successful in propagating HNSCC cells. This study included a high proportion of patients from underrepresented minority groups. LEVEL OF EVIDENCE: Not Applicable Laryngoscope, 134:2748-2756, 2024.

Low-level expression of miR-375 correlates with poor outcome and metastasis while altering the invasive properties of head and neck squamous cell carcinomas.

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

CDK4/6 Inhibition Induces Senescence and Enhances Radiation Response by Disabling DNA Damage Repair in Oral Cavity Squamous Cell Carcinoma.

PURPOSE: HPV(-) OCSCC resists radiation treatment. The CDKN2A gene, encoding p16INK4A, is commonly disrupted in OCSCC. p16 inhibits CDK4/CDK6, leading to cell cycle arrest, but the biological sequelae of CDK4/6 inhibition in OCSCC remains understudied. This study examines whether inhibition of CDK4/6 enhances radiation response in OCSCC. METHODS: MTT assays were performed in OCSCC cell lines HN5 and CAL27 following treatment with palbociclib. Clonogenic survival and synergy were analyzed after radiation (RT-2 or 4Gy), palbociclib (P) (0.5 µM or 1 µM), or concurrent combination treatment (P+RT). DNA damage/repair and senescence were examined. CDK4/6 were targeted via siRNA to corroborate P+RT effects. Three-dimensional immortalized spheroids and organoids derived from patient tumors (conditionally reprogrammed OCSCC CR-06 and CR-18) were established to further examine and validate responses to P+RT. RESULTS: P+RT demonstrated reduced viability and synergy, increased ß-gal expression (~95%), and ~two-fold higher γH2AX. Rad51 and Ku80 were reduced after P+RT, indicating impairment of both HR and NHEJ. siCDK4/6 increased senescence with radiation. Spheroids showed reduced proliferation and size with P+RT. CR-06 and CR-18 further demonstrated three-fold reduced proliferation and organoids size with P+RT. CONCLUSION: Targeting CDK4/6 can lead to improved efficacy when combined with radiation in OCSCC by inducing senescence and inhibiting DNA damage repair.

A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer.

Detection of human papillomavirus (HPV) in head and neck cancer has therapeutic implications. In situ hybridization and immunohistochemistry for p16 are used by surgical pathologists. We compared the sensitivity and specificity of three popular commercial tests for HPV detection in head and neck squamous cell carcinomas with a 'gold standard' HPV PCR assay. A total of 110 prospectively collected, formalin-fixed tumor specimens were compiled onto tissue microarrays and tested for HPV DNA by in situ hybridization with two probe sets, a biotinylated probe for high-risk (HR) HPV types 16/18 (Dako, CA, USA) and a probe cocktail for 16/18, plus 10 additional HR types (Ventana, AZ, USA). The p16(INK4) expression was also assessed using a Pharmingen immunohistochemistry antibody (BD Biosciences, CA, USA). Tissue microarrays were stained and scored at expert laboratories. HPV DNA was detected by MY09/11-PCR, using Gold AmpliTaq and dot-blot hybridization on matched-fresh frozen specimens in a research laboratory. HPV 16 E6 and E7-RNA expression was also measured using RT-PCR. Test performance was assessed by a receiver operating characteristic analysis. HR-HPV DNA types 16, 18 and 35 were detected by MY-PCR in 28% of tumors, with the majority (97%) testing positive for type 16. Compared with MY-PCR, the sensitivity and specificity for HR-HPV DNA detection with Dako in situ hybridization was 21% (95% confidence interval (CI): 7-42) and 100% (95% CI: 93-100), respectively. Corresponding test results by Ventana in situ hybridization were 59% (95% CI: 39-78) and 58% (95% CI: 45-71), respectively. The p16 immunohistochemistry performed better overall than Dako (P=0.042) and Ventana (P=0.055), with a sensitivity of 52% (95% CI: 32-71) and specificity of 93% (95% CI: 84-98). Compared with a gold standard HPV-PCR assay, HPV detection by in situ hybridization was less accurate for head and neck squamous cell carcinoma on tissue microarrays than p16 immunohistochemistry. Further testing is warranted before these assays should be recommended for clinical HPV detection.

Low-level expression of microRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cell carcinoma.

Small noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. The importance of miRNAs as potential cancer prognostic indicators is underscored by their involvement in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) tumor and adjacent normal tissue were examined by microarray analysis and validated by quantitative TaqMan real-time polymerase chain reaction. Using TaqMan real-time polymerase chain reaction we measured the quantitative associations between a subset of miRNAs identified on microarrays in primary tumors at diagnosis and cancer survival in a cohort of 104 HNSCC patients undergoing treatment with curative intent. The majority of miRNAs exhibiting altered expression in primary human HNSCC tumors (including miR-1, miR-133a, miR-205, and let-7d) show lower expression levels relative to normal adjacent tissue. In contrast, hsa-miR-21 is frequently overexpressed in human HNSCC tumors. Using univariate and multivariable statistical models we show that low levels of hsa-miR205 are significantly associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. In addition, combined low levels of hsa-miR-205 and hsa-let-7d expression in HNSCC tumors are significantly associated with poor head and neck cancer survival Our results show that miRNA expression levels can be used as prognostic markers of head and neck cancer.

The histone deacetylase inhibitor LBH589 inhibits expression of mitotic genes causing G2/M arrest and cell death in head and neck squamous cell carcinoma cell lines.

Head and neck squamous cell carcinoma represents a complex set of neoplasms arising in diverse anatomical locations. The site and stage of the cancer determine whether patients will be treated with single or multi-modality therapy. The HDAC inhibitor LBH589 is effective in treating some haematological neoplasms and shows promise for certain epithelial neoplasms. As with other human cancer cell lines, LBH589 causes up-regulation of p21, G2/M cell cycle arrest, and cell death of human HNSCC cell lines, as measured using flow cytometry and cDNA microarrays. Global RNA expression studies following treatment of the HNSCC cell line FaDu with LBH589 reveal down-regulation of genes required for chromosome congression and segregation (SMC2L1), sister chromatid cohesion (DDX11), and kinetochore structure (CENP-A, CENP-F, and CENP-M); these LBH589-induced changes in gene expression coupled with the down-regulation of MYC and BIRC5 (survivin) provide a plausible explanation for the early mitotic arrest and cell death observed. When LBH589-induced changes in gene expression were compared with gene expression profiles of 41 primary HNSCC samples, many of the genes that were down-regulated by LBH589 showed increased expression in primary HNSCC, suggesting that some patients with HNSCC may respond to treatment with LBH589.

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma.

OBJECTIVES: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. METHODS: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. RESULTS: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. CONCLUSION: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2643-2649, 2020.

Optimal targeting of BCL-family proteins in head and neck squamous cell carcinoma requires inhibition of both BCL-xL and MCL-1.

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares - Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9µM (range 6.6µM - 13.9µM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7µM (range, 8.8µM to 12.7µM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.

Proteomic analysis of oral cavity squamous cell carcinoma specimens identifies patient outcome-associated proteins.

CONTEXT: Global proteomic analysis of oral cavity squamous cell carcinoma was performed to identify changes that reflect patient outcomes. OBJECTIVES: To identify differentially expressed proteins associated with patient outcomes and to explore the use of imaging mass spectrometry as a clinical tool to identify clinically relevant proteins. DESIGN: Two-dimensional separation of digested peptides generated from 43 specimens with high-resolution mass spectrometry identified proteins associated with disease-specific death, distant metastasis, and loco-regional recurrence. RNA expressions had been correlated to protein levels to test transcriptional regulation of clinically relevant proteins. Imaging mass spectrometry explored an alternative platform for assessing clinically relevant proteins that would complement surgical pathologic diagnosis. RESULTS: Seventy-two peptide features were found to be associated with 3 patient outcomes: disease-specific death (9), distant metastasis (16), and loco-regional recurrence (39); 8 of them were associated with multiple outcomes. Functional ontology revealed major changes in cell adhesion and calcium binding. Thirteen RNAs showed strong correlation with their encoded proteins, implying transcriptional control. Reduction of DSP, PKP1, and TRIM29 was associated with significantly shorter time to onset of distant metastasis. Reduction of PKP1 and TRIM29 correlated with poorer disease-specific survival. Additionally, S100A8 and S100A9 reductions were verified for their association with poor prognosis using imaging mass spectrometry, a platform more adaptable for use with surgical pathology. CONCLUSIONS: Using global proteomic analysis, we have identified proteins associated with clinical outcomes. The list of clinically relevant proteins observed will provide a means to develop clinical assays for prognosis and optimizing treatment selection.

Human papillomavirus-associated head and neck squamous cell carcinoma survival: a comparison by tumor site and initial treatment.

Recent evidence suggests that human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) patients have better survival than HPV-negative patients. However, it is unclear if similar patterns for survival exist across different tumor sites, and whether HPV-associated prognosis is modified by type of treatment. We prospectively tested 222 histologically confirmed HNSCC primary tumors for HPV DNA by PCR and HPV E6/E7 RNA by RT-PCR prior to treatment at a large urban health center. Cox proportional hazard ratio models were constructed to assess HPV-associated differences in overall and disease-specific survival adjusting for clinical and demographic covariates. HPV detection varied significantly by primary HNSCC tumor site, from 35 % for oropharynx, to 25 % for hypopharynx, 5 % for larynx, and 3 % for oral cavity (p < 0.0001), with HPV16 accounting for the majority (95 %) of HPV-positive tumors. The hazard-risk of overall and disease-specific death comparing HPV16-positive versus negative oropharyngeal HNSCC was reduced by 74 and 89 %, respectively (p values < 0.05), and was independent of other prognostic indicators; no statistically significant changes in outcomes were observed for non-oropharyngeal HNSCC sites. Prediction of overall survival was better with combined DNA and RNA HPV16 positive PCR detection. There was no difference in HPV16-associated survival whether patients received either surgery or (chemo)radiotherapy as their initial treatment modality. Improved HPV-associated HNSCC survival is limited to patients with oropharyngeal primaries. No selective treatment advantage is observed for HPV-positive tumors, although clinical trials are needed to evaluate which treatment modalities provide the most benefit for HPV-positive HNSCC.

Inhibition of Plk1 and Cyclin B1 expression results in panobinostat-induced G2 delay and mitotic defects.

The development of clinically useful histone deacetylase inhibitors has expanded greatly. In a preclinical study, we showed that panobinostat (LBH589) inhibits cell cycle progression of human head and neck squamous cell carcinoma (HNSCC) cell lines at G2/M and an associated decrease in expression of particular genes required for passage through G2 and mitosis. In this study we sought to analyse the mechanistic underpinnings of panobinostat-induced growth arrest. HNSCC cell lines were synchronised and progression through mitosis monitored. We demonstrate that panobinostat causes a marked G2 delay and mitotic defects. A loss of G2-specific Plk1 and Cyclin B1 expression and co-incident increase in p21(Waf1/Cip1) expression is also shown. Furthermore, we show a significant loss of E2F1 recruitment to the promoters of these genes in response to panobinostat treatment. These data provide mechanistic evidence of panobinostat-induced cell cycle arrest and highlight its potential as a chemotherapeutic agent for HNSCC.

Cytoplasmic ezrin and moesin correlate with poor survival in head and neck squamous cell carcinoma.

Members of the 4.1 superfamily of proteins, including ezrin, moesin, merlin, and willin regulate many normal physiologic processes such as cellular shape, motility, and proliferation. In addition, they contribute both to tumor development and tumor progression. We reported previously that strong cytoplasmic ezrin expression was independently associated with poorer patient survival. One hundred and thirty-one histologically confirmed primary head and neck squamous cell carcinomas were examined prospectively for cancer progression and survival at a large health care center in the Bronx, NY, USA. Immunohistochemical analysis of ezrin, moesin, merlin, and willin expression in tissue microarray samples of primary head and neck squamous cell carcinoma revealed a significant association of increased cytoplasmic ezrin with poor cancer survival. Global RNA analyses suggest that cancers with high cytoplasmic ezrin have a more invasive phenotype. This study supports our previous findings associating cytoplasmic ezrin with more aggressive behavior and poorer outcome and indicates the need for a multi-institutional study to validate the use of cytoplasmic ezrin as a biomarker for treatment planning in head and neck squamous cell carcinoma.

Site-specific molecular signatures predict aggressive disease in HNSCC.

It is known that head and neck squamous cell carcinomas (HNSCC) originating from different anatomic locations can exhibit varying behavior that is not predictable by histopathology of the primary tumor. Using a microarray containing 27,323 cDNA clones, we generated sets of gene expression profiles for 36 HNSCC primary tumors (12 oral cavity, 12 oropharynx, and 12 larynx/hypopharynx). From these datasets, we ranked genes according to their ability to differentiate between patients whose disease progressed within a 24 month period (aggressive phenotype) and those that did not (non-aggressive phenotype) based on levels of gene expression. A merging of datasets from the three sites revealed that only a fraction of identified genes were shared between any two sites. This contrasted greatly with the significant overlap (approximately 50%) in down-regulated genes identified in tumor/normal comparisons using cases both from oropharynx and larynx/hypopharynx. From these data, we conclude that HNSCC tumors originating from different anatomic sites share consistent changes in gene expression when comparing primary tumors to normal adjacent mucosa; these common changes most likely reflect alterations required for tumor development. In contrast, once a tumor has developed, tumor-host interactions at the different anatomic sites are likely responsible for the site-specific signatures associated with aggressive versus non-aggressive disease. Predictions of outcome based on gene expression profiling are therefore heavily influenced by the anatomic site of the primary tumor.

Global proteomic analysis distinguishes biologic differences in head and neck squamous carcinoma.

The goal of this study was to establish a method for detecting biologically significant differences in protein expression of head and neck squamous cell carcinoma (HNSCC) obtained from the same samples utilized in gene expression analyses. Proteins from two head and neck tumor cell lines, SCC-25 and FaDu, were isolated from the denatured protein solution remaining from the TRIzol extraction procedure used for isolation of total RNA for microarray analysis. Peptides resulting from chemical and enzymatic digestion of the proteins were first separated by strong cation-exchange chromatography, followed by liquid chromatography-mass spectrometry (LC-MS) analysis on a QqTOF mass spectrometer. Stable isotope-labeled synthetic peptides were added to each ion-exchange fraction as internal standards, for reversed-phase HPLC retention time alignment. Protein extraction and digestion were repeated three times for each cell line and each extract was analyzed three times by LC-MS. To discriminate between technical vs biological variation, the ion-exchange fraction, retention time, normalized mass and signal intensity of these nine data sets were constructed into numerical arrays for statistical analysis. Of the approximately 50,000 signals, 90 peptide ions were found to discriminate the two cell lines with high stringency. Of those, six peptides were derived from vimentin and four peptides were derived from annexin II; both expressed more in SCC-25. Follow-up analysis of some of these signals by LC-MS/MS and RNA expression profiling revealed both concordance and discordance of RNA and protein expression. This study demonstrates that this procedure is highly reliable for identifying peptides that distinguish biological variability among samples, indicating that this method can be applied to study clinical samples, to identify potential prognostic biomarkers for HNSCC.

Deficiency in the anti-apoptotic protein A1-a results in a diminished acute inflammatory response.

A1 is an anti-apoptotic member of the Bcl-2 family that is up-regulated in inflammatory myeloid cells. In the present study, we investigated the role of A1 in the maintenance of acute inflammation in mice. Mice possess three genes encoding highly related isoforms of A1. A1-a isoform mRNA was minimally expressed in resident peritoneal macrophages, but was present at a 300-fold higher level in inflammatory macrophages elicited by i.p. infection with Toxoplasma gondii. In comparison, A1-b and A1-d levels were 3- and 10-fold higher, respectively. Peritoneal leukocytosis was decreased in infected A1-a-deficient mice compared with wild-type, and this reduction was associated with a small but reproducible enhancement of survival. These effects could not be explained by an alteration in peritoneal parasite load, nor by increased apoptosis of infected inflammatory cells, which were protected from cell death by an A1-a-independent mechanism. Increased apoptosis in inflammatory neutrophils was observed sporadically in A1-a-deficient mice. Regulation of apoptosis by A1-a may be an important proinflammatory event in acute host responses.