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Determination of malignancy in thyroid nodules remains a major diagnostic challenge. Here we report the feasibility and clinical utility of developing an AI-defined protein-based biomarker panel for diagnostic classification of thyroid nodules: based initially on formalin-fixed paraffin-embedded (FFPE), and further refined for fine-needle aspiration (FNA) tissue specimens of minute amounts which pose technical challenges for other methods. We first developed a neural network model of 19 protein biomarkers based on the proteomes of 1724 FFPE thyroid tissue samples from a retrospective cohort. This classifier achieved over 91% accuracy in the discovery set for classifying malignant thyroid nodules. The classifier was externally validated by blinded analyses in a retrospective cohort of 288 nodules (89% accuracy; FFPE) and a prospective cohort of 294 FNA biopsies (85% accuracy) from twelve independent clinical centers. This study shows that integrating high-throughput proteomics and AI technology in multi-center retrospective and prospective clinical cohorts facilitates precise disease diagnosis which is otherwise difficult to achieve by other methods., (© 2022. The Author(s).)

Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess tumor-initiating (TIC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of TIC phenotypes. Many of the implicated GPCRs signal through the G protein GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in patients. GNA13 levels modulate drug resistance and TIC-like phenotypes in patient-derived head and neck squamous cell carcinoma (HNSCC) cells in vitro and in vivo. Blockade of GNA13 expression, or of select downstream pathways, using small-molecule inhibitors abrogates GNA13-induced TIC phenotypes, rendering cells vulnerable to standard-of-care cytotoxic therapies. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker for tumor progression, and that interfering with GNA13-induced signaling provides a novel strategy to block TICs and drug resistance in HNSCCs.

Although Kruppel-like factor 5 (KLF5) is a transcription factor that has been implicated in pathways critical to carcinogenesis, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Here, we describe a novel role for KLF5 in a p53-independent apoptotic pathway. Using RNA-interference technology, we show that cells deficient in KLF5 have increased sensitivity to DNA damage, regardless of p53 status. Both p53 and p53-dependent factors are unaffected by KLF5 depletion. Instead, the apoptotic phenotype consequent to damage is associated with reduced bad phosphorylation, and downregulation of Pim1. Consistently, transfection of wild-type Pim1 is sufficient to rescue this phenotype. Previous data have shown a number of putative Sp1-binding consensus sequences on the Pim1 promoter. Remarkably, chromatin immunoprecipitation studies show that KLF5 binds to the Pim1 promoter, and that binding increases soon after damage. These results identify a novel, p53-independent apoptotic pathway through which KLF5 functions in response to DNA damage. Therapeutic deregulation of this pathway could be used to modulate chemosensitivity.

We analysed 148 primary breast cancers using BAC-arrays containing 287 clones representing cancer-related gene/loci to obtain genomic molecular portraits. Gains were detected in 136 tumors (91.9%) and losses in 123 tumors (83.1%). Eight tumors (5.4%) did not have any genomic aberrations in the 281 clones analysed. Common (more than 15% of the samples) gains were observed at 8q11-qtel, 1q21-qtel, 17q11-q12 and 11q13, whereas common losses were observed at 16q12-qtel, 11ptel-p15.5, 1p36-ptel, 17p11.2-p12 and 8ptel-p22. Patients with tumors registering either less than 5% (median value) or less than 11% (third quartile) total copy number changes had a better overall survival (log-rank test: P=0.0417 and P=0.0375, respectively). Unsupervised hierarchical clustering based on copy number changes identified four clusters. Women with tumors from the cluster with amplification of three regions containing known breast oncogenes (11q13, 17q12 and 20q13) had a worse prognosis. The good prognosis group (Nottingham Prognostic Index (NPI)

The role of the transcriptional coactivator p300 in cell cycle control has not been analysed in detail due to the lack of appropriate experimental systems. We have now examined cell cycle progression of p300-deficient cancer cell lines, where p300 was disrupted either by gene targeting (p300(-) cells) or knocked down using RNAi. Despite significant proliferation defects under normal growth conditions, p300-deficient cells progressed rapidly through G1 with premature S-phase entry. Accelerated G1/S transition was associated with early retinoblastoma (RB) hyperphosphorylation and activation of E2F targets. The p300-acetylase activity was dispensable since expression of a HAT-deficient p300 mutant reversed these changes. Co-immunoprecipitation showed p300/RB interaction occurs in vivo during G1, and this interaction has two peaks: in early G1 with unphosphorylated RB and in late G1 with phosphorylated RB. In vitro kinase assays showed that p300 directly inhibits cdk6-mediated RB phosphorylation, suggesting p300 acts in early G1 to prevent RB hyperphosphorylation and delay premature S-phase entry. Paradoxically, continued cycling of p300(-) cells despite prolonged serum depletion was observed, and this occurred in association with persistent RB hyperphosphorylation. Altogether, these results suggest that p300 has an important role in G1/S control, possibly by modulating RB phosphorylation.

p300 and cyclic AMP response element-binding protein (CBP) are adenoviral E1A-binding proteins involved in multiple cellular processes, and function as transcriptional co-factors and histone acetyltransferases. Germline mutation of CBP results in Rubinstein-Taybi syndrome, which is characterized by an increased predisposition to childhood malignancies. Furthermore, somatic mutations of p300 and CBP occur in a number of malignancies. Chromosome translocations target CBP and, less commonly, p300 in acute myeloid leukemia and treatment-related hematological disorders. p300 mutations in solid tumors result in truncated p300 protein products or amino-acid substitutions in critical protein domains, and these are often associated with inactivation of the second allele. A mouse model confirms that p300 and CBP function as suppressors of hematological tumor formation. The involvement of these proteins in critical tumorigenic pathways (including TGF-beta, p53 and Rb) provides a mechanistic route as to how their inactivation could result in cancer.