Nuclear factor-κB (NF-κB) signaling contributes to human disease processes, notably inflammatory diseases and cancer. NF-κB has a role in tumorigenesis and tumor growth, as well as promotion of ...metastases. Mechanisms responsible for abnormal NF-κB activation are not fully elucidated; however, RelA phosphorylation, particularly at serine residues S536 and S276, is critical for RelA function. Kinases that phosphorylate RelA promote oncogenic behaviors, suggesting that phosphatases targeting RelA could have tumor-inhibiting activities; however, few RelA phosphatases have been identified. Here, we identified tumor inhibitory and RelA phosphatase activities of the protein phosphatase 2C (PP2C) phosphatase family member, PPM1A. We show that PPM1A directly dephosphorylated RelA at residues S536 and S276 and selectively inhibited NF-κB transcriptional activity, resulting in decreased expression of monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 and interleukin-6, cytokines implicated in cancer metastasis. PPM1A depletion enhanced NF-κB-dependent cell invasion, whereas PPM1A expression inhibited invasion. Analyses of human expression data revealed that metastatic prostate cancer deposits had lower PPM1A expression compared with primary tumors without distant metastases. A hematogenous metastasis mouse model revealed that PPM1A expression inhibited bony metastases of prostate cancer cells after vascular injection. In summary, our findings suggest that PPM1A is a RelA phosphatase that regulates NF-κB activity and that PPM1A has tumor suppressor-like activity. Our analyses also suggest that PPM1A inhibits prostate cancer metastases and as neither gene deletions nor inactivating mutations of PPM1A have been described, increasing PPM1A activity in tumors represents a potential therapeutic strategy to inhibit NF-κB signaling or bony metastases in human cancer.
High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head ...and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.
Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by ...sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC.
Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR.
Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation.
Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs.
Human papilloma viruses (HPV) are linked to an epidemic increase in oropharyngeal head and neck squamous cell carcinomas (HNSCC), which display viral inactivation of tumor suppressors TP53 and RB1 ...and rapid regional spread. However, the role of genomic alterations in enabling the modulation of pathways that promote the aggressive phenotype of these cancers is unclear. Recently, a subset of HPV
HNSCC has been shown to harbor novel genetic defects or decreased expression of
(
). TRAF3 has been implicated as a negative regulator of alternative NF-κB pathway activation and activator of antiviral type I IFN response to other DNA viruses. How
alterations affect pathogenesis of HPV
HNSCC has not been extensively investigated. Here, we report that TRAF3-deficient HPV
tumors and cell lines exhibit increased expression of alternative NF-κB pathway components and transcription factors NF-κB2/RELB. Overexpression of
in HPV
cell lines with decreased endogenous TRAF3 inhibited NF-κB2/RELB expression, nuclear localization, and NF-κB reporter activity, while increasing the expression of IFNA1 mRNA and protein and sensitizing cells to its growth inhibition. Overexpression of TRAF3 also enhanced TP53 and RB tumor suppressor proteins and decreased HPV E6 oncoprotein in HPV
cells. Correspondingly, TRAF3 inhibited cell growth, colony formation, migration, and resistance to TNFα and cisplatin-induced cell death. Conversely,
knockout enhanced colony formation and proliferation of an HPV
HNSCC line expressing higher TRAF3 levels. Together, these findings support a functional role of
as a tumor suppressor modulating established cancer hallmarks in HPV
HNSCC.
These findings report the functional role of TRAF3 as a tumor suppressor that modulates the malignant phenotype of HPV
head and neck cancers.
.
Background
NUT carcinoma is an aggressive malignancy characterized by translocations in the NUTM1 gene. There are currently no consensus treatment recommendations for NUT carcinomas.
Methods
Here, we ...describe the case of a previously healthy male diagnosed with NUT carcinoma after presenting with sinus pressure, found to have a sinonasal mass and distant metastatic disease in the lungs. While pathologic evaluation and immunohistochemistry were consistent with NUT carcinoma, initial genomic profiling did not demonstrate a NUTM1 translocation.
Results
Whole transcriptomic RNA sequencing of the tumor revealed a YAP1‐NUTM1 fusion. Based on an in vitro drug sensitivity screen, the patient was treated with gemcitabine and nab‐paclitaxel, achieving a partial response that persisted for 9 months.
Conclusions
Unbiased transcriptomic sequencing may identify previously uncharacterized NUTM1 fusion partners. Gemcitabine and nab‐paclitaxel is a well‐tolerated combination chemotherapy regimen and could offer a novel treatment approach for NUT carcinoma.
Unbiased transcriptome sequencing may identify previously uncharacterized NUTM1 fusion partners. Gemcitabine and nab‐paclitaxel is a well‐tolerated combination chemotherapy regimen and could offer a novel treatment approach for NUT carcinoma.
Cancer stem cells (CSC) are considered the major cause of aggressive tumor behavior, recurrence, metastases, and resistance to radiation, making them an attractive therapeutic target. However, ...isolation of CSC from tumor tissue and their characterization are challenging due to uncertainty about their molecular markers and conditions for their propagation. Adenoid cystic carcinoma (ACC), which arises predominantly in the salivary glands, is a slow-growing but relentless tumor that frequently invades nerves and metastasizes. New effective treatment approaches for ACC have not emerged over the last 40years. Previously, based on a highly conserved SOX10 gene signature that we identified in the majority of ACC tumors, we suggested the existence in ACC of SOX10+ cells with neural stem properties and corroborated this hypothesis via isolation from ACC tissue a novel population of CSC, termed ACC-CSC. These cells activated NOTCH1 signaling and co-expressed SOX10 and other ACC-intrinsic neural crest stem cell markers with CD133, a CSC cell surface marker, suggesting that ACC is driven by a previously uncharacterized population of SOX10+/CD133+ cells with neural stem cell properties. Here, we authenticated ACC identity of our primary cultures by demonstrating that most of them harbor MYB-NFIB fusions, which are found in 86% of ACC. We demonstrated using CyTOF, a novel mass cytometry technology, that these cells express high β-catenin and STAT3 levels and are marked by CD24 and CD44. Finally, to streamline development of ACC cell lines, we developed RT-PCR tests for distinguishing mouse and human cells and used immunomagnetic cell sorting to eliminate mouse cells from long-term cell cultures. Overall, this study describes a new population of CSC that activates signaling pathways associated with poor prognosis, validates their ACC identity, and optimizes approaches that can be used for purification of ACC-CSC and generation of cell lines.
•ACC cancer stem cell cultures are authenticated using ACC-intrinsic MYB fusions.•A novel MYB fusion, with a long non-coding RNA from 6q23.4, is identified.•ACC-CSC is marked by expression of CD24 and CD44.•ACC-CSC purification is optimized to establish cell lines for drug screening.•CyTOF is used to explore STAT3 and β-catenin as novel targets for ACC-CSC eradication.
Data are limited on the prognostic value of human papillomavirus (HPV) status for head and neck carcinoma subsites.
To determine whether HPV positivity at each head and neck subsite is associated ...with improved overall survival.
This retrospective population-based cohort study used the National Cancer Database to identify patients diagnosed with head and neck squamous cell carcinomas from January 1, 2010, to December 31, 2014. Patients were classified according to the location of their primary malignancy into 1 of the 6 main subsites of the upper aerodigestive tract: oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, and sinonasal tract. Patients were also classified by their HPV status. Data collection for this study took place from January 1, 2010, to December 31, 2014. Data analysis was conducted from August 1, 2017, to September 30, 2017.
The difference in 5-year overall survival between patients with HPV-positive status and those with HPV-negative status in various head and neck carcinoma subsites; the role of HPV status in an unadjusted Cox multivariate regression model.
Of the 175 223 total number of patients identified (129 634 74.0% male; 45 589 26.0% female; mean SD age, 63.1 11.9 years), 133 273 (76.1%) were ineligible and 41 950 (23.9%) were included in the sample. This sample included 16 644 patients (39.7%) with HPV-positive tumors and 25 306 (60.3%) with HPV-negative tumors. Patients with an HPV-positive status were more likely to be younger, be white, be male, present with local T category tumors, and have poor differentiation on histologic examination. HPV-positive status was associated with survival at 4 tumor subsites: oral cavity (hazard ratio HR, 0.76; 95% CI, 0.66-0.87), oropharynx (HR, 0.44; 95% CI, 0.41-0.47), hypopharynx (HR, 0.59; 95% CI, 0.45-0.77), and larynx (HR, 0.71; 95% CI, 0.59-0.85). The HPV status was the greatest factor in survival outcome between the HPV-positive and -negative cohorts at the oropharynx subsite (77.6% vs 50.7%; survival difference, 26.9%; 95% CI, 25.6%-28.2%) and hypopharynx subsites (52.2% vs 28.8%; survival difference, 23.4%; 95% CI, 17.5%-29.3%). For the nasopharynx (HR, 1.03; 95% CI, 0.75-1.42) and sinonasal tract (HR, 0.63; 95% CI, 0.39-1.01) subsites, HPV-positive status was not an independent prognostic factor.
Human papillomavirus positivity was associated with improved survival in 4 subsites (oropharynx, hypopharynx, oral cavity, and larynx), and the largest survival difference was noted in the oropharynx and hypopharynx subsites. In the nasopharynx and sinonasal tract subsites, HPV positivity had no association with overall survival. Given these results, routine testing for HPV at the oropharynx, hypopharynx, oral cavity, and larynx subsites may be warranted.
To conduct a retrospective review of 168 consecutively treated locally advanced head-and-neck cancer (LAHNC) patients treated with intensity-modulated radiotherapy (IMRT)/chemotherapy, to determine ...the rate and risk factors for developing hypothyroidism.
Intensity-modulated radiotherapy was delivered in 33 daily fractions to 69.3 Gy to gross disease and 56.1 Gy to clinically normal cervical nodes. Dose-volume histograms (DVHs) of IMRT plans were used to determine radiation dose to thyroid and were compared with DVHs using conventional three-dimensional radiotherapy (3D-RT) in 10 of these same patients randomly selected for replanning and with DVHs of 16 patients in whom the thyroid was intentionally avoided during IMRT. Weekly paclitaxel (30 mg/m(2)) and carboplatin area under the curve-1 were given concurrently with IMRT.
Sixty-one of 128 evaluable patients (47.7%) developed hypothyroidism after a median of 1.08 years after IMRT (range, 2.4 months to 3.9 years). Age and volume of irradiated thyroid were associated with hypothyroidism development after IMRT. Compared with 3D-RT, IMRT with no thyroid dose constraints resulted in significantly higher minimum, maximum, and median dose (p < 0.0001) and percentage thyroid volume receiving 10, 20, and 60 Gy (p < 0.05). Compared with 3D-RT, IMRT with thyroid dose constraints resulted in lower median dose and percentage thyroid volume receiving 30, 40, and 50 Gy (p < 0.005) but higher minimum and maximum dose (p < 0.005).
If not protected, IMRT for LAHNC can result in higher radiation to the thyroid than with conventional 3D-RT. Techniques to reduce dose and volume of radiation to thyroid tissue with IMRT are achievable and recommended.
LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss ...of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.
Highlights • Regulation of RelA dephosphorylation is important for restraining RelA activity. • Two families of phosphatases have been identified as direct RelA phosphatases. • The tumor suppressor, ...LZAP, is associated with dephosphorylation of RelA and regulating PPM family members. • Proteins that accelerate RelA dephosphorylation display tumor suppressor-like activities.
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