Pure estrogen receptor antagonists potentiate capecitabine activity in ESR1-mutant breast cancer.

The ESR1 ligand binding domain activating mutations are the most prevalent genetic mechanism of acquired endocrine resistance in metastatic hormone receptor-positive breast cancer. These mutations confer endocrine resistance that remains estrogen receptor (ER) dependent. We hypothesized that in the presence of the ER mutations, continued ER blockade with endocrine therapies that target mutant ER is essential for tumor suppression even with chemotherapy treatment. Here, we conducted comprehensive pre-clinical in vitro and in vivo experiments testing the efficacy of adding fulvestrant to fluorouracil (5FU) and the 5FU pro-drug, capecitabine, in models of wild-type (WT) and mutant ER. Our findings revealed that while this combination had an additive effect in the presence of WT-ER, in the presence of the Y537S ER mutation there was synergy. Notably, these effects were not seen with the combination of 5FU and selective estrogen receptor modulators, such as tamoxifen, or in the absence of intact P53. Likewise, in a patient-derived xenograft (PDX) harboring a Y537S ER mutation the addition of fulvestrant to capecitabine potentiated tumor suppression. Moreover, multiplex immunofluorescence revealed that this effect was due to decreased cell proliferation in all cells expressing ER and was not dependent on the degree of ER expression. Taken together, these results support the clinical investigation of the combination of ER antagonists with capecitabine in patients with metastatic hormone receptor-positive breast cancer who have experienced progression on endocrine therapy and targeted therapies, particularly in the presence of an ESR1 activating mutation.

Combined PIK3CA and SOX2 Gene Amplification Predicts Laryngeal Cancer Risk beyond Histopathological Grading.

The PIK3CA and SOX2 genes map at 3q26, a chromosomal region frequently amplified in head and neck cancers, which is associated with poor prognosis. This study explores the clinical significance of PIK3CA and SOX2 gene amplification in early tumorigenesis. Gene copy number was analyzed by real-time PCR in 62 laryngeal precancerous lesions and correlated with histopathological grading and laryngeal cancer risk. Amplification of the SOX2 and PIK3CA genes was frequently detected in 19 (31%) and 32 (52%) laryngeal dysplasias, respectively, and co-amplification in 18 (29%) cases. The PIK3CA and SOX2 amplifications were predominant in high-grade dysplasias and significantly associated with laryngeal cancer risk beyond histological criteria. Multivariable Cox analysis further revealed PIK3CA gene amplification as an independent predictor of laryngeal cancer development. Interestingly, combined PIK3CA and SOX2 amplification allowed us to distinguish three cancer risk subgroups, and PIK3CA and SOX2 co-amplification was found the strongest predictor by ROC analysis. Our data demonstrate the clinical relevance of PIK3CA and SOX2 amplification in early laryngeal tumorigenesis. Remarkably, PIK3CA amplification was found to be an independent cancer predictor. Furthermore, combined PIK3CA and SOX2 amplification is emerging as a valuable and easy-to-implement tool for cancer risk assessment in patients with laryngeal precancerous lesions beyond current WHO histological grading.

Tumor mutational burden predictability in head and neck squamous cell carcinoma patients treated with immunotherapy: systematic review and meta-analysis.

BACKGROUND: Tumor mutational burden (TMB) has been demonstrated to predict the response to immune checkpoint inhibitors (ICIs) in various cancers. However, the role of TMB in head and neck squamous cell carcinoma (HNSCC) has not yet been specifically addressed. Since HNSCC patients exhibit a rather limited response to ICIs, there is an unmet need to develop predictive biomarkers to improve patient selection criteria and the clinical benefit of ICI treatment. METHODS: We conducted a systematic review and meta-analysis according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines. HNSCC cohort studies were selected when TMB prior to ICI treatment was evaluated, TMB cutoff value was available, and the prognostic value of TMB was evaluated by time-to-event survival analysis. A total of 11 out of 1960 articles were analyzed, including 1200 HNSCC patients. RESULTS: The results showed that those patients harboring high TMB exhibited a significantly superior overall response rate (OR = 2.62; 95% CI 1.74-3.94; p < 0.0001) and a survival advantage (HR = 0.53; 95% CI 0.39-0.71; p < 0.0001) after ICI treatment. CONCLUSION: This is the first meta-analysis to demonstrate a higher response and clinical benefit from ICI therapy in HNSCC patients with high TMB.

Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor-positive Breast Cancer.

PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.

Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor-Positive Breast Cancer.

Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated ß2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of ß2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer. SIGNIFICANCE: Adding SMAC mimetics to endocrine therapy enhances tumor regression in a cell autonomous manner while increasing tumor immunogenicity, indicating that this combination could be an effective treatment for HR+ patients with breast cancer.

Targeting oncogenic functions of miR-301a in head and neck squamous cell carcinoma by PI3K/PTEN and MEK/ERK pathways.

Treatment of head and neck squamous cell carcinomas (HNSCC), the sixth most frequent cancer worldwide, remains challenging. miRNA dysregulation is closely linked to tumorigenesis and tumor progression, thus emerging as suitable targets for cancer treatment. Transcriptomic analysis of TCGA HNSCC dataset revealed that miR-301a expression levels significantly increased in primary tumors, as compared to patient-matched normal tissue. This prompted us to investigate its pathobiological role and potential as new therapeutic target using different preclinical HNSCC models. miR-301a overexpression in HNSCC-derived cell lines led to enhanced proliferation and invasion, whereas miR-301 inhibition reduced these effects. In vivo validation was performed using an orthotopic mouse model. Results concordantly showed that the mitotic counts, the percentage of infiltration depth and Ki67 proliferative index were significantly augmented in the subgroup of mice harboring miR-301a-overexpressing tumors. Further mechanistic characterization revealed PI3K/PTEN/AKT and MEK/ERK pathways as central signaling nodes responsible for mediating the oncogenic activity of miR-301a observed in HNSCC cells. Notably, pharmacological disruption of PI3K and ERK signals with BYL-719 and PD98059, respectively, was effective to completely revert/abolish miR-301a-promoted tumor cell growth and invasion. Altogether, these findings demonstrate that miR-301a dysregulation plays an oncogenic role in HNSCC, thus emerging as a candidate therapeutic target for this disease. Importantly, available PI3K and ERK inhibitors emerge as promising anti-tumor agents to effectively target miR-301a-mediated signal circuit hampering growth-promoting and pro-invasive functions.

Driving role of head and neck cancer cell secretome on the invasion of stromal fibroblasts: Mechanistic insights by phosphoproteomics.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are major players in tumor-stroma communication, and participate in several cancer hallmarks to drive tumor progression and metastatic dissemination. This study investigates the driving effects of tumor-secreted factors on CAF biology, with the ultimate goal of identifying effective therapeutic targets/strategies for head and neck squamous cell carcinomas (HNSCC). METHODS: Functionally, conditioned media (CM) from different HNSCC-derived cell lines and normal keratinocytes (Kc) were tested on the growth and invasion of populations of primary CAFs and normal fibroblasts (NFs) using 3D invasion assays in collagen matrices. The changes in MMPs expression were evaluated by RT-qPCR and kinase enrichment was analyzed using mass spectrometry phosphoproteomics. RESULTS: Our results consistently demonstrate that HNSCC-secreted factors (but not Kc CM) specifically and robustly promoted pro-invasive properties in both CAFs and NFs, thereby reflecting the plasticity of fibroblast subtypes. Concomitantly, HNSCC-secreted factors massively increased metalloproteinases levels in CAFs and NFs. By contrast, HNSCC CM and Kc CM exhibited comparable growth-promoting effects on stromal fibroblasts. Mechanistically, phosphoproteomic analysis predominantly revealed phosphorylation changes in fibroblasts upon treatment with HNSCC CM, and various promising kinases were identified: MKK7, MKK4, ASK1, RAF1, BRAF, ARAF, COT, PDK1, RSK2 and AKT1. Interestingly, pharmacologic inhibition of RAF1/BRAF using sorafenib emerged as the most effective drug to block tumor-promoted fibroblast invasion without affecting fibroblast viability CONCLUSIONS: Our findings demonstrate that HNSCC-secreted factors specifically fine tune the invasive potential of stromal fibroblasts, thereby generating tumor-driven pro-invasive niches, which in turn to ultimately facilitate cancer cell dissemination. Furthermore, the RAF/BRAF inhibitor sorafenib was identified as a promising candidate to effectively target the onset of pro-invasive clusters of stromal fibroblasts in the HNSCC microenvironment.

Pathobiological functions and clinical implications of annexin dysregulation in human cancers.

Annexins are an extensive superfamily of structurally related calcium- and phospholipid-binding proteins, largely conserved and widely distributed among species. Twelve human annexins have been identified, referred to as Annexin A1-13 (A12 remains as of yet unassigned), whose genes are spread throughout the genome on eight different chromosomes. According to their distinct tissue distribution and subcellular localization, annexins have been functionally implicated in a variety of biological processes relevant to both physiological and pathological conditions. Dysregulation of annexin expression patterns and functions has been revealed as a common feature in multiple cancers, thereby emerging as potential biomarkers and molecular targets for clinical application. Nevertheless, translation of this knowledge to the clinic requires in-depth functional and mechanistic characterization of dysregulated annexins for each individual cancer type, since each protein exhibits varying expression levels and phenotypic specificity depending on the tumor types. This review specifically and thoroughly examines the current knowledge on annexin dysfunctions in carcinogenesis. Hence, available data on expression levels, mechanism of action and pathophysiological effects of Annexin A1-13 among different cancers will be dissected, also further discussing future perspectives for potential applications as biomarkers for early diagnosis, prognosis and molecular-targeted therapies. Special attention is devoted to head and neck cancers (HNC), a complex and heterogeneous group of aggressive malignancies, often lately diagnosed, with high mortality, and scarce therapeutic options.

A Distinct Chromatin State Drives Therapeutic Resistance in Invasive Lobular Breast Cancer.

Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor-positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1-estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1-ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1-ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. SIGNIFICANCE: A unique FOXA1-ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668.

The ESR1 Mutations: From Bedside to Bench to Bedside.

The ESR1 ligand-binding mutations were unveiled a number of years ago and are the most common genetic mechanism of acquired resistance to endocrine treatment, particularly, to aromatase inhibitors. The discovery of these mutations was enabled after advancements in sequencing technologies and when metastatic tissue samples were interrogated. The ESR1 ligand-binding domain mutations are activating mutations that lead to constitutive ligand-independent activity, which explains the emergence of these mutations under the selective pressure of aromatase inhibitors. Arnesen and colleagues have generated new models of the ESR1 mutations using CRISPR technology to generate single-cell-derived clones in which the ESR1 ligand-binding mutations were "knocked-in" and expressed under the endogenous promoter of estrogen receptor. The authors have extensively characterized these models and have shed new light on the functional consequences ESR1 mutations.See related article by Arnesen et al., p. 539.

Tumor-Infiltrating CD20+ B Lymphocytes: Significance and Prognostic Implications in Oral Cancer Microenvironment.

Immunohistochemical analysis of stromal/tumoral CD20+ B lymphocytes was performed in 125 OSCC patients. Correlations with immune profiles CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocytes (TILs), tumoral PD-L1, and stem-related factors NANOG and SOX2 were assessed, and also associations with clinical data and patient survival. There was a strong positive correlation between the infiltration of CD20+ B lymphocytes and other immune profiles (i.e., CD4+, CD8+, and FOXP3+ TILs, and CD68+ and CD163+ macrophages) both in stroma and tumor nests. Strikingly, CD20+ TILs were inversely correlated with NANOG/SOX2 expression. Stromal CD20+ TILs were significantly associated with T classification and second primary tumors. A stratified survival analysis showed that tumoral CD20+ TILs were significantly associated with prognosis in male and younger patients, with tobacco or alcohol consumption, high tumoral CD8+ TILs, low tumoral infiltration by CD68+ macrophages, positive PD-L1 expression, and negative NANOG/SOX2. Multivariate Cox analysis further revealed clinical stage and tumoral CD20+ TILs independently associated with disease-specific survival (HR = 2.42, p = 0.003; and HR = 0.57, p = 0.04, respectively). In conclusion, high CD20+ TIL density emerges as an independent good prognostic factor in OSCC, suggesting a role in antitumor immunity. This study also uncovered an inverse correlation between CD20+ TILs and CSC marker expression.

Lectin-Like Transcript 1 (LLT1) Checkpoint: A Novel Independent Prognostic Factor in HPV-Negative Oropharyngeal Squamous Cell Carcinoma.

Lectin-like transcript 1 (LLT1) expression by tumor cells contributes to immune evasion, thereby emerging as a natural killer (NK) cell-mediated immunotherapeutic target. This study is the first to investigate LLT1 expression (encoded by CLEC2D gene) in head and neck cancers to ascertain its impact on patient prognosis. LLT1 expression was analyzed by immunohistochemistry in a homogeneous cohort of human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinomas (OPSCC), and correlated with clinical data. Results were further validated using transcriptomic data from the TCGA database. Tumoral LLT1 expression was detected in 190/221 (86%) OPSCC specimens, whereas normal pharyngeal epithelium was negative. Patients harboring LLT1-positive tumors showed significantly lower disease-specific (DSS) and overall survival (OS) (p = 0.049 and p = 0.036, respectively, log-rank test). High density of LLT1-positive tumor-infiltrating lymphocytes (TIL) was also frequently detected in 160 (73%) OPSCC samples, and significantly associated with better DSS and OS (p < 0.001 and p = 0.007, respectively). Multivariate Cox analysis further revealed that tumoral LLT1 expression and infiltration of LLT1-positive TIL were independent prognostic factors for DSS and OS. CLEC2D mRNA levels are also significantly increased in primary tumors compared to normal tissue. Strikingly, the prognostic impact of CLEC2D mRNA levels varied depending on HPV status in OPSCC, and among distinct cancer types. CLEC2D expression was significantly correlated with NK cell infiltration using the MCP-counter model. These findings uncover LLT1/CLEC2D as an independent prognostic factor in HPV-negative OPSCC, and a potential novel target for immunotherapy.

Impact of notch signaling on the prognosis of patients with head and neck squamous cell carcinoma.

OBJECTIVES: The function of NOTCH signaling (oncogenic or oncosuppressive) remains controversial in head and neck squamous cell carcinomas (HNSCC). The purpose of this work is to investigate the role of NOTCH pathway in HNSCC prognosis. METHODS: Immunohistochemical NOTCH1 and HES1 expression was jointly evaluated and correlated with other NOTCH1 targets, p21 (WAF1/Cip1) and Cyclin D1, using an unbiased cohort of 372 surgically treated HPV-negative HNSCC patients. RESULTS: Membranous NOTCH1 expression was detected in 197 (61%) out of 324 evaluable tumor samples, and nuclear NOTCH1 expression in 91 samples (28%). Nuclear HES1 expression was found in 224 (67%) cases. Membranous and nuclear NOTCH1 expression were consistently and significantly correlated with nuclear HES1 (P < 0.001) and p21 (P = 0.03) expression, but not with Cyclin D1. NOTCH1 expression was significantly associated to early stages (I-II), non-recurrent disease, and better disease-specific (DSS) and overall survival (OS) rates (P < 0.001). Moreover, triple-positive cases (NOTCH1+/HES1+/p21+) exhibited significantly improved DSS (P < 0.001) and OS (P = 0.004), thus reinforcing the association of NOTCH pathway activation with a better prognosis in HNSCC. Multivariate analysis further revealed membranous NOTCH1 expression as a robust independent predictor of better DSS (HR = 0.554; 95% IC 0.412-0.745; P < 0.001) and better OS (HR = 0.640; 95% CI 0.491-0.835; P = 0.001). CONCLUSION: These findings show the association of NOTCH pathway activation with a better prognosis in HNSCC patients, also revealing membranous NOTCH1 expression as a robust independent predictor of improved survival. Accordingly, these results suggest a tumor suppressive rather than an oncogenic role for NOTCH pathway in HNSCC.

Prognostic Significance of the Pluripotency Factors NANOG, SOX2, and OCT4 in Head and Neck Squamous Cell Carcinomas.

Cancer stem cells (CSCs) play major roles in tumor initiation, progression, and resistance to cancer therapy. Several CSC markers have been studied in head and neck squamous cell carcinomas (HNSCC), including the pluripotency factors NANOG, SOX2, and OCT4; however, their clinical significance is still unclear. NANOG, SOX2, and OCT4 expression was evaluated by immunochemistry in 348 surgically-treated HNSCC, and correlated with clinicopathological parameters and patient outcomes. mRNA expression was further analyzed in 530 The Cancer Genome Atlas (TCGA) HNSCC. NANOG protein expression was detected in 250 (72%) cases, more frequently in patients with lymph node metastasis (p = 0.003), and was an independent predictor of better survival in multivariate analysis. While OCT4 expression was undetectable, SOX2 expression was observed in 105 (30%) cases, and strongly correlated with NANOG expression. Combined expression of both proteins showed the highest survival rates, and double-negative cases the worst survival. Strikingly, the impact of NANOG and SOX2 on outcome varied depending on tumor site and lymph node infiltration, specifically showing prognostic significance in pharyngeal tumors. Correlation between NANOG and SOX2 at mRNA and protein was specifically observed in node positive (N+) patients, and consistently correlated with better survival rates. According to our findings, NANOG protein expression is frequent in HNSCC, thereby emerging as an independent predictor of better prognosis in pharyngeal tumors. Moreover, this study uncovers a differential impact of NANOG and SOX2 expression on HNSCC prognosis, depending on tumor site and lymph node infiltration, which could facilitate high-risk patient stratification.

Expression of E-cadherin and ß-catenin in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas. / Expresión de E-cadherina y ß-catenina en carcinomas de células escamosas de laringe e hipofaringe.

INTRODUCTION AND OBJECTIVES: Dysfunction of the E-cadherin/catenin complex is directly related to carcinogenesis and metastases development. The aim of this paper is to investigate the prognostic significance of E-cadherin and ß-catenin expression in surgically treated laryngeal and hypopharyngeal squamous cell carcinomas. MATERIAL AND METHODS: Tumour tissue samples were obtained from 133 consecutive patients with squamous cell carcinomas of the head and neck: 68 of the larynx and 65 hypopharyngeal carcinomas, who underwent surgical treatment in our hospital between 2000 and 2005. E-cadherin and ß-catenin expression was analysed by immunohistochemistry, quantifying the percentage of stained cells and the intensity of staining. RESULTS: E-cadherin and ß-catenin expression was evaluable in 59 laryngeal carcinomas and in 58 cases of hypopharyngeal carcinomas. In the laryngeal tumours, a significant association was found between the low expression of membrane ß-catenin with T4 tumours and tumour recurrence. In the hypopharynx there was a significant association between positive expression of nuclear ß-catenin and poor histological differentiation (P=.02). In the multivariate analysis, only the presence of lymph node metastases was an independent predictive factor of decreased disease-specific survival in laryngeal squamous cell carcinomas. CONCLUSIONS: The expression of E-cadherin and ß-catenin does not show prognostic significance in laryngeal and hypopharyngeal squamous cell carcinomas over the TNM classification.

The Differential Impact of SRC Expression on the Prognosis of Patients with Head and Neck Squamous Cell Carcinoma.

Aberrant SRC expression and activation is frequently detected in multiple cancers, and hence, targeting SRC has emerged as a promising therapeutic strategy. Different SRC inhibitors have demonstrated potent anti-tumor activity in preclinical models, although they largely lack clinical efficacy as monotherapy in late-stage solid tumors, including head and neck squamous cell carcinomas (HNSCC). Adequate selection and stratification of patients who may respond to and benefit from anti-SRC therapies is therefore needed to guide clinical trials and treatment efficacy. This study investigates the prognostic significance of active SRC expression in a homogeneous cohort of 122 human papillomavirus (HPV)-negative, surgically treated HNSCC patients. Immunohistochemical evaluation of the active form of SRC by means of anti-SRC Clone 28 monoclonal antibody was specifically performed and subsequently correlated with clinical data. The expression of p-SRC (Tyr419), total SRC, and downstream SRC effectors was also analyzed. Our results uncovered striking differences in the prognostic relevance of SRC expression in HNSCC patients depending on the tumor site. Active SRC expression was found to significantly associate with advanced disease stages, presence of lymph node metastasis, and tumor recurrences in patients with laryngeal tumors, but not in the pharyngeal subgroup. Multivariate Cox analysis further revealed active SRC expression as an independent predictor of cancer-specific mortality in patients with laryngeal carcinomas. Concordantly, expression of p-SRC (Tyr419) and the SRC substrates focal adhesion kinase (FAK) and the Arf GTPase-activating protein ASAP1 also showed specific associations with poor prognosis in the larynx. These findings could have important implications in ongoing Src family kinase (SFK)-based clinical trials, as these new criteria could help to improve patient selection and develop biomarker-stratified trials.

SOX2 Expression Is an Independent Predictor of Oral Cancer Progression.

Potentially malignant oral lesions, mainly leukoplakia, are common. Malignant transformation varies widely, even in the absence of histological features such as dysplasia. Hence, there is a need for novel biomarker-based systems to more accurately predict the risk of cancer progression. The pluripotency transcription factor SOX2 is frequently overexpressed in cancers, including oral squamous cell carcinoma (OSCC), thereby providing a link between malignancy and stemness. This study investigates the clinical relevance of SOX2 protein expression in early stages of oral carcinogenesis as a cancer risk biomarker, and also its impact on prognosis and disease outcome at late stages of OSCC progression. SOX2 expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and in 125 patients with OSCC, and correlated with clinicopathological data and outcomes. Nuclear SOX2 expression was detected in four (7%) cases of oral epithelial dysplasia, using a cut-off of 10% stained nuclei, and in 16 (29%) cases when any positive nuclei was evaluated. Univariate analysis showed that SOX2 expression and histopathological grading were significantly associated with oral cancer risk; and both were found to be significant independent predictors in the multivariate analysis. Nuclear SOX2 expression was also found in 49 (39%) OSCC cases, was more frequent in early tumor stages and N0 cases, and was associated with a better survival. In conclusion, SOX2 expression emerges as an independent predictor of oral cancer risk in patients with oral leukoplakia. These findings underscore the relevant role of SOX2 in early oral tumorigenesis rather than in tumor progression.

The SRC Inhibitor Dasatinib Induces Stem Cell-Like Properties in Head and Neck Cancer Cells that are Effectively Counteracted by the Mithralog EC-8042.

The frequent dysregulation of SRC family kinases (SFK) in multiple cancers prompted various inhibitors to be actively tested in preclinical and clinical trials. Disappointingly, dasatinib and saracatinib failed to demonstrate monotherapeutic efficacy in patients with head and neck squamous cell carcinomas (HNSCC). Deeper functional and mechanistic knowledge of the actions of these drugs is therefore needed to improve clinical outcome and to develop more efficient combinational strategies. Even though the SFK inhibitors dasatinib and saracatinib robustly blocked cell migration and invasion in HNSCC cell lines, this study unveils undesirable stem cell-promoting functions that could explain the lack of clinical efficacy in HNSCC patients. These deleterious effects were targeted by the mithramycin analog EC-8042 that efficiently eliminated cancer stem cells (CSC)-enriched tumorsphere cultures as well as tumor bulk cells and demonstrated potent antitumor activity in vivo. Furthermore, combination treatment of dasatinib with EC-8042 provided favorable complementary anti-proliferative, anti-invasive, and anti-CSC functions without any noticeable adverse interactions of both agents. These findings strongly support combinational strategies with EC-8042 for clinical testing in HNSCC patients. These data may have implications on ongoing dasatinib-based trials.

Deciphering the Molecular Basis of Melatonin Protective Effects on Breast Cells Treated with Doxorubicin: TWIST1 a Transcription Factor Involved in EMT and Metastasis, a Novel Target of Melatonin.

Melatonin mitigates cancer initiation, progression and metastasis through inhibition of both the synthesis of estrogens and the transcriptional activity of the estradiol-ER (Estrogen receptor) complex in the estrogen-dependent breast cancer cell line MCF-7. Moreover, melatonin improves the sensitivity of MCF-7 to chemotherapeutic agents and protects against their side effects. It has been described that melatonin potentiates the anti-proliferative effects of doxorubicin; however, the molecular changes involving gene expression and the activation/inhibition of intracellular signaling pathways remain largely unknown. Here we found that melatonin enhanced the anti-proliferative effect of doxorubicin in MCF-7 but not in MDA-MB-231 cells. Strikingly, doxorubicin treatment induced cell migration and invasion, and melatonin effectively counteracted these effects in MCF-7 but not in estrogen-independent MDA-MB-231 cells. Importantly, we describe for the first time the ability of melatonin to downregulate TWIST1 (Twist-related protein 1) in estrogen-dependent but not in estrogen-independent breast cancer cells. Combined with doxorubicin, melatonin inhibited the activation of p70S6K and modulated the expression of breast cancer, angiogenesis and clock genes. Moreover, melatonin regulates the levels of TWIST1-related microRNAs, such as miR-10a, miR-10b and miR-34a. Since TWIST1 plays a pivotal role in the epithelial to mesenchymal transition, acquisition of metastatic phenotype and angiogenesis, our results suggest that inhibition of TWIST1 by melatonin might be a crucial mechanism of overcoming resistance and improving the oncostatic potential of doxorubicin in estrogen-dependent breast cancer cells.

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.