HPV16 genome structure analysis in oropharyngeal cancer PDXs identifies tumors with integrated and episomal genomes.

HPV + oropharyngeal squamous cell carcinoma (OPC) incidence recently surpassed cervical cancer and is the most common HPV-related cancer in the developed world. HPV16 is in ∼90 % of HPV + OPCs, with episomal genomes in the majority of cases. Most existing HPV16+ cancer cell lines derive from outside the oropharynx and harbor integrated HPV genomes. Thus, there is need for OPC preclinical models to evaluate standard and experimental therapeutics in the presence of episomal HPV16 oncogenic drivers. Here we characterize HPV genome structures in eight HPV16+ OPC patient-derived xenografts (PDXs), and evaluate their responses to standard chemotherapy. HPV genome state was investigated by combining Southern blot, T5 exonuclease assay, whole genome sequencing, and RNAseq data. This analysis revealed complexity and variation in integrated vs. episomal HPV forms across PDXs and demonstrated that four PDXs predominantly contain episomal HPV16. Episomal status did not ensure favorable in vivo responses to cisplatin therapy, despite the more favorable prognosis previously attributed to episomal HPV + tumors; this could be due to the small number present in the dataset. Our analysis establishes PDX models as test platforms for novel therapies designed to target maintenance of the episomal forms of HPV16 that commonly appear in OPC.

A novel pipeline for prioritizing cancer type-specific therapeutic vulnerabilities using DepMap identifies PAK2 as a target in head and neck squamous cell carcinomas.

There is limited guidance on exploiting the genome-wide loss-of-function CRISPR screens in cancer Dependency Map (DepMap) to identify new targets for individual cancer types. This study integrated multiple tools to filter these data in order to seek new therapeutic targets specific to head and neck squamous cell carcinoma (HNSCC). The resulting pipeline prioritized 143 targetable dependencies that represented both well-studied targets and emerging target classes like mitochondrial carriers and RNA-binding proteins. In total, 14 targets had clinical inhibitors used for other cancers or nonmalignant diseases that hold near-term potential to repurpose for HNSCC therapy. Comparing inhibitor response data that were publicly available for 13 prioritized targets between the cell lines with high vs. low dependency on each target uncovered novel therapeutic potential for the PAK2 serine/threonine kinase. PAK2 gene dependency was found to be associated with wild-type p53, low PAK2 mRNA, and diploid status of the 3q amplicon containing PAK2. These findings establish a generalizable pipeline to prioritize clinically relevant targets for individual cancer types using DepMap. Its application to HNSCC highlights novel relevance for PAK2 inhibition and identifies biomarkers of PAK2 inhibitor response.

HPV E6 regulates therapy responses in oropharyngeal cancer by repressing the PGC-1α/ERRα axis.

Therapy with radiation plus cisplatin kills HPV+ oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why these standard treatments fail for some patients, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs affects mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlations. Ectopically expressing fl-E6 in models with low baseline levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the peroxisome proliferator-activated receptor gamma co-activator 1α/estrogen-related receptor α (PGC-1α/ERRα) pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1α transcription. Concordant observations were made in 3 clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, the highest p53 target gene expression, and an activated PGC-1α/ERRα pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively affecting patient survival. E6's interaction with the PGC-1α/ERRα axis has implications for predicting and targeting treatment resistance in OPSCC.

The strong propensity of Cadherin-23 for aggregation inhibits cell migration.

Cadherin-23 (Cdh23), a long-chain non-classical cadherin, exhibits strong homophilic and heterophilic binding. The physiological relevance of strong heterophilic binding with protocadherin-15 at neuroepithelial tip links is well-studied. However, the role of Cdh23 homodimers in physiology is less understood, despite its widespread expression at the cell boundaries of various human and mouse tissues, including kidney, muscle, testes, and heart. Here, we performed immunofluorescence studies that revealed that Cdh23 is present as distinct puncta at the cell-cell boundaries of cancer cells. Analysis of patient data and quantitative estimation of Cdh23 in human tissues (normal and tumor) also indicated that Cdh23 is down-regulated via promoter methylation in lung adenocarcinoma (AD) and esophageal squamous cell carcinoma (SCC) cells; we also observed a clear inverse correlation between Cdh23 expression and cancer metastasis. Using HEK293T cells and four types of cancer cells differentially expressing Cdh23, we observed that cell migration was faster in cells with reduced levels of Cdh23 expression. The cell migration rate in cancer cells is further accelerated by the presence of excretory isoforms of Cdh23, which loosen its cell-adhesion ability by competitive binding. Overall, our data indicate the role of Cdh23 as a suppressor of cell migration.

Cellulose-metallothionein matrix for metal binding.

In this report, we have modified bacterial cellulose to a metal binding matrix by covalently conjugating physiological metal chelators known as metallothioneins. The hydroxyl groups of the native bacterial cellulose from Gluconobacter xylinus are epoxidized, followed by the covalent conjugation with the amine groups of the proteins. For the first time, a covalent conjugation of protein with bacterial cellulose is achieved using the epoxy-amine conjugation chemistry. Using this protocol, 50% mass by mass of the metallothionein could be attached to bacterial cellulose. The morphological features and porosity of the modified cellulose are different compared to pristine bacterial cellulose. Also, the conjugated material has better thermal stability. A five-fold enhancement in the metal binding capacity of the metallothionein conjugated bacterial cellulose is achieved as compared to pristine bacterial cellulose. Cellular metabolic assay and membrane integrity assay on MCF and HeLa cell lines showed no significant toxicity of the conjugate material. This bacterial cellulose-metallothionein conjugate can be explored for health care applications where management of metal toxicity is crucial. Further, the epoxy-amine chemistry for covalent conjugation of protein can be applied for several other types of proteins to develop specific functional biocompatible and biodegradable cellulose matrices.