RUNX transcription factors are essential in maintaining epididymal epithelial differentiation.

Apart from the androgen receptor, transcription factors (TFs) that are required for the development and formation of the different segments of the epididymis have remained unknown. We identified TF families expressed in the developing epididymides, of which many showed segment specificity. From these TFs, down-regulation of runt related transcription factors (RUNXs) 1 and 2 expression coincides with epithelial regression in Dicer1 cKO mice. Concomitant deletion of both Runx1 and Runx2 in a mouse epididymal epithelial cell line affected cell morphology, adhesion and mobility in vitro. Furthermore, lack of functional RUNXs severely disturbed the formation of 3D epididymal organoid-like structures. Transcriptomic analysis of the epididymal cell organoid-like structures indicated that RUNX1 and RUNX2 are involved in the regulation of MAPK signaling, NOTCH pathway activity, and EMT-related gene expression. This suggests that RUNXs are master regulators of several essential signaling pathways, and necessary for the maintenance of proper differentiation of the epididymal epithelium.

The effect of matrices on the gene expression profile of patient-derived head and neck carcinoma cells for in vitro therapy testing.

OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with a 5-year mortality rate of ~ 50%. New in vitro methods are needed for testing patients' cancer cell response to anti-cancer treatments. We aimed to investigate how the gene expression of fresh carcinoma tissue samples and freshly digested single cancer cells change after short-term cell culturing on plastic, Matrigel or Myogel. Additionally, we studied the effect of these changes on the cancer cells' response to anti-cancer treatments. MATERIALS/METHODS: Fresh tissue samples from HNSCC patients were obtained perioperatively and single cells were enzymatically isolated and cultured on either plastic, Matrigel or Myogel. We treated the cultured cells with cisplatin, cetuximab, and irradiation; and performed cell viability measurement. RNA was isolated from fresh tissue samples, freshly isolated single cells and cultured cells, and RNA sequencing transcriptome profiling and gene set enrichment analysis were performed. RESULTS: Cancer cells obtained from fresh tissue samples changed their gene expression regardless of the culturing conditions, which may be due to the enzymatic digestion of the tissue. Myogel was more effective than Matrigel at supporting the upregulation of pathways related to cancer cell proliferation and invasion. The impacts of anti-cancer treatments varied between culturing conditions. CONCLUSIONS: Our study showed the challenge of in vitro cancer drug testing using enzymatic cell digestion. The upregulation of many targeted pathways in the cultured cells may partially explain the common clinical failure of the targeted cancer drugs that pass the in vitro testing.

Preparing biomedical students for the unknown: Some unusual challenges for students to help them understand the fundamentals of empirical research.

Make teaching challenging again: a pedagogic approach to educate students on how to do science rather than learn about it.

Increased expression of fibroblast growth factor 13 in prostate cancer is associated with shortened time to biochemical recurrence after radical prostatectomy.

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high-risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co-labeled cells suggested a possible interaction of FGF13 with α-tubulin and the voltage-gated sodium channel proteins (Na(V)s/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR-free survival, in particular if combined with other clinical parameters.

Validation of proposed prostate cancer biomarkers with gene expression data: a long road to travel.

Biomarkers are important for early detection of cancer, prognosis, response prediction, and detection of residual or relapsing disease. Special attention has been given to diagnostic markers for prostate cancer since it is thought that early detection and surgery might reduce prostate cancer-specific mortality. The use of prostate-specific antigen, PSA (KLK3), has been debated on the base of cohort studies that show that its use in preventive screenings only marginally influences mortality from prostate cancer. Many groups have identified alternative or additional markers, among which PCA3, in order to detect early prostate cancer through screening, to distinguish potentially lethal from indolent prostate cancers, and to guide the treatment decision. The large number of markers proposed has led us to the present study in which we analyze these indicators for their diagnostic and prognostic potential using publicly available genomic data. We identified 380 markers from literature analysis on 20,000 articles on prostate cancer markers. The most interesting ones appeared to be claudin 3 (CLDN3) and alpha-methysacyl-CoA racemase highly expressed in prostate cancer and filamin C (FLNC) and keratin 5 with highest expression in normal prostate tissue. None of the markers proposed can compete with PSA for tissue specificity. The indicators proposed generally show a great variability of expression in normal and tumor tissue or are expressed at similar levels in other tissues. Those proposed as prognostic markers distinguish cases with marginally different risk of progression and appear to have a clinically limited use. We used data sets sampling 152 prostate tissues, data sets with 281 prostate cancers analyzed by microarray analysis and a study of integrated genomics on 218 cases to develop a multigene score. A multivariate model that combines several indicators increases the discrimination power but does not add impressively to the information obtained from Gleason scoring. This analysis of 10 years of marker research suggests that diagnostic and prognostic testing is more difficult in prostate cancer than in other neoplasms and that we must continue to search for better candidates.

Targeting the cancer cells and cancer-associated fibroblasts with next-generation FGFR inhibitors in prostate cancer co-culture models.

BACKGROUND: Inhibition of androgen receptor (AR) signaling is the main treatment strategy in advanced prostate cancer (PCa). A subset of castration resistant prostate cancer (CRPC) bypasses the AR blockade by increased fibroblast growth factor receptor (FGFR) signaling. The first- and second-generation, non-covalent FGFR inhibitors (FGFRis) have largely failed in the clinical trials against PCa. PURPOSE: In this study, we tested the drug sensitivity of LNCaP, VCaP, and CWR-R1PCa cell lines to second-generation, covalent FGFRis (FIIN1, FIIN2) and a novel FGFR downstream molecule inhibitor (FRS2αi). METHODS: 2D and 3D mono- and co-cultures of cancer cells, and cancer-associated fibroblasts (CAFs) were used to mimic tumor-stroma interactions in the extracellular matrix (ECM). The treatment responses of the FGFR signaling molecules, the viability and proliferation of cancer cells, and CAFs were determined through immunoblotting, migration assay, cell viability assay, and real-time imaging. Immunofluorescent and confocal microscopy images of control and treated cultures of cancer cells and CAFs, and their morphometric data were deduced. RESULTS: The FGFRis were more effective in mono-cultures of the cancer cells compared with co-cultures with CAFs. The FRS2αi was specifically effective in co-cultures with CAFs but was not cytotoxic to CAF mono-cultures as in the case of FIIN1 and FIIN2. At the molecular level, FRS2αi decreased p-FRS2α, p-ERK1/2, and activated apoptosis as monitored by cleaved caspase-3 activity in a concentration-dependent manner in the co-cultures. We observed no synergistic drug efficacy in the combination treatment of the FGFRi with ARi, enzalutamide, and darolutamide. The FRS2αi treatment led to a decrease in proliferation of cancer cell clusters in co-cultures as indicated by their reduced size and Ki67 expression. CONCLUSIONS: CAFs exert a protective effect on cancer cells and should be included in the in vitro models to make them physiologically more relevant in screening and testing of FGFRis. The FRS2αi was the most potent agent in reducing the viability and proliferation of the 3D organotypic co-cultures, mainly by disrupting the contact between CAFs and cancer cell clusters. The next-generation FGFRi, FRS2αi, may be a better alternative treatment option for overcoming ARi treatment resistance in advanced PCa.

The Notch inhibitor, FLI-06, increases the chemosensitivity of head and neck Squamous cell carcinoma cells to taxanes-based treatment.

Aberration of Notch signaling is one of the key events involved in the development and progression of head and neck squamous cell carcinoma (HNSCC). The Notch pathway controls the tissue-specific differentiation of normal squamous epithelial cells and is frequently altered in squamous carcinomas, thus affecting their proliferation, growth, survival, and chemosensitivity or resistance against anti-cancer agents. In this study, we show that the use of novel, small-molecule inhibitors of Notch signaling, such as FLI-06, can have a beneficial effect on increasing the chemosensitivity of HNSCC to taxane-based chemotherapy. Inhibition of Notch signaling by FLI-06 alone virtually blocks the proliferation and growth of HNSCC cells in both 2D and 3D cultures and the zebrafish model, which is accompanied by down-regulation of key Notch target genes and proteins. Mechanistically, FLI-06 treatment causes cell cycle arrest in the G1-phase and induction of apoptosis in HNSCC, which is accompanied by increased c-JunS63 phosphorylation. Combining FLI-06 with Docetaxel shows a synergistic effect and partially blocks the cell growth of aggressive HNSCC cells via enhanced apoptosis and modification of c-JunS243 phosphorylation via GSK-3ß inhibition. In conclusion, inhibition of Notch signaling in HNSCC cells that retain active Notch signaling significantly supports taxane-based anticancer activities via modulation of both the GSK-3ß and the c-Jun.

Assessment of targeted therapy opportunities in sinonasal cancers using patient-derived functional tumor models.

Malignant tumors derived from the epithelium lining the nasal cavity region are termed sinonasal cancers, a highly heterogeneous group of rare tumors accounting for 3 - 5 % of all head and neck cancers. Progress with next-generation molecular profiling has improved our understanding of the complexity of sinonasal cancers and resulted in the identification of an increasing number of distinct tumor entities. Despite these significant developments, the treatment of sinonasal cancers has hardly evolved since the 1980s, and an advanced sinonasal cancer presents a poor prognosis as targeted therapies are usually not available. To gain insights into potential targeted therapeutic opportunities, we performed a multiomics profiling of patient-derived functional tumor models to identify molecular characteristics associated with pharmacological responses in the different subtypes of sinonasal cancer. METHODS: Patient-derived ex vivo tumor models representing four distinct sinonasal cancer subtypes: sinonasal intestinal-type adenocarcinoma, sinonasal neuroendocrine carcinoma, sinonasal undifferentiated carcinoma and SMARCB1 deficient sinonasal carcinoma were included in the analyses. Results of functional drug screens of 160 anti-cancer therapies were integrated with gene panel sequencing and histological analyses of the tumor tissues and the ex vivo cell cultures to establish associations between drug sensitivity and molecular characteristics including driver mutations. RESULTS: The different sinonasal cancer subtypes display considerable differential drug sensitivity. Underlying the drug sensitivity profiles, each subtype was associated with unique molecular features. The therapeutic vulnerabilities correlating with specific genomic background were extended and validated with in silico analyses of cancer cell lines representing different human cancers and with reported case studies of sinonasal cancers treated with targeted therapies. CONCLUSION: The results demonstrate the importance of understanding the differential biology and the molecular features associated with the different subtypes of sinonasal cancers. Patient-derived ex vivo tumor models can be a powerful tool for investigating these rare cancers and prioritizing targeted therapeutic strategies for future clinical development and personalized medicine.

Differentiation-promoting culture of competent and noncompetent keratinocytes identifies biomarkers for head and neck cancer.

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

Modulation of Notch Signaling by Small-Molecular Compounds and Its Potential in Anticancer Studies.

Notch signaling is responsible for conveying messages between cells through direct contact, playing a pivotal role in tissue development and homeostasis. The modulation of Notch-related processes, such as cell growth, differentiation, viability, and cell fate, offer opportunities to better understand and prevent disease progression, including cancer. Currently, research efforts are mainly focused on attempts to inhibit Notch signaling in tumors with strong oncogenic, gain-of-function (GoF) or hyperactivation of Notch signaling. The goal is to reduce the growth and proliferation of cancer cells, interfere with neo-angiogenesis, increase chemosensitivity, potentially target cancer stem cells, tumor dormancy, and invasion, and induce apoptosis. Attempts to pharmacologically enhance or restore disturbed Notch signaling for anticancer therapies are less frequent. However, in some cancer types, such as squamous cell carcinomas, preferentially, loss-of-function (LoF) mutations have been confirmed, and restoring but not blocking Notch functions may be beneficial for therapy. The modulation of Notch signaling can be performed at several key levels related to NOTCH receptor expression, translation, posttranslational (proteolytic) processing, glycosylation, transport, and activation. This further includes blocking the interaction with Notch-related nuclear DNA transcription. Examples of small-molecular chemical compounds, that modulate individual elements of Notch signaling at the mentioned levels, have been described in the recent literature.

Optimization of a Three-Dimensional Culturing Method for Assessing the Impact of Cisplatin on Notch Signaling in Head and Neck Squamous Cell Carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized "3D sheet model" approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.

Evaluation of the anticancer activity of RIN-1, a Notch signaling modulator, in head and neck squamous cell carcinoma.

Notch signalling is one of the key molecular pathways involved in cell-to-cell signal transduction. Although the mechanisms of action of the NOTCH receptors are already relatively well known, their biological implications remain unclear, especially during the initiation and progression of head and neck squamous cell carcinoma (HNSCC). Here, we present the growth- and differentiation-modulating effects of various "next generation" small molecule Notch modulators represented by RIN-1, and CB-103, on HNSCC, compared to gamma secretase inhibitors as "conventional" NOTCH interfering compounds, like DAPT. These molecules were tested in different cell- and tissue culture conditions represented by 2D monolayer, non-adherent or spheroid culture, 3D organoid cultures, and zebrafish in vivo model. The most pronounced, pleiotropic effects were observed for the NOTCH modulator RIN-1. At the molecular level, RIN-1-dependent activation of Notch signalling led to characteristic changes in the expression of NOTCH-regulated targets, i.e., the transcriptional suppressors HES1 and HEY1, p21 (CDKN1A) cell cycle inhibitor, and pro-apoptotic BAX markers. These changes led to restriction of proliferation, growth, and reduced motility of HNSCC cells in 2D cultures. Consequently, cell cycle arrest in the G2-M phase and induction of apoptosis were observed. Similar anticancer effects were observed in 3D cultures and in the zebrafish model. In contrast, RIN-1 treatment resulted in inhibition of Notch signalling and the growth of HNSCC spheroids under non-adherent cell culture conditions. Our results suggest that modulation of Notch signalling could be used as a chemotherapeutic agent in selected patients with intact NOTCH signaling.

Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression.

Fibroblast growth factor receptors (FGFRs) 1-4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor-stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

A cell spot microarray method for production of high density siRNA transfection microarrays.

BACKGROUND: High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible. RESULTS: Here, we describe the optimization of a miniaturized cell spot microarray (CSMA) method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells. CONCLUSIONS: The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.

Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis.

Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions.

Proteomic analysis of field cancerization in pharynx and oesophagus: a prospective pilot study.

'Field cancerization' in head and neck squamous cell carcinoma (HNSCC) is poorly understood and it may extend from the pharynx into the oesophagus. Both local recurrences and second primary carcinomas/second field tumours may originate from field cancerization. Our prospective pilot study aimed at the identification of patients suffering from field cancerization on the basis of mucosal protein profiles. Five mucosal biopsies from the oropharynx, hypopharynx and from three regions of the oesophagus were taken from 24 patients. Protein profiles were generated from the mucosal biopsies. After classifier learning, using the profiles of the patients without tumour diagnosis (n = 9), we were able to discriminate between the different mucosal sites and between healthy mucosa and HNSCC using tumour and healthy tissue samples. Mucosal biopsies of tumour patients (n = 15) revealed changes in the protein profiles similar to those in the tumours. During 42 months median follow-up, six tumour patients experienced local recurrences and second field tumours, of which three occurred in the oesophagus. In all six cases, tumour relapse was correctly predicted by altered mucosal protein profiles (p = 0.007, Fisher's exact test, two-tailed). Consequently, molecular field cancerization had a strong impact on progression-free survival (p = 0.007, log-rank test). Protein profiles of small diagnostic biopsies hold great promise to improve personalized risk assessment in HNSCC. Larger studies are needed to further substantiate these findings.

CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer.

CD73 is a cell surface ecto-5'-nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5'-(α, ß-methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial-mesenchymal transition.

Shooting at Moving and Hidden Targets-Tumour Cell Plasticity and the Notch Signalling Pathway in Head and Neck Squamous Cell Carcinomas.

Head and Neck Squamous Cell Carcinoma (HNSCC) is often aggressive, with poor response to current therapies in approximately 40-50% of the patients. Current therapies are restricted to operation and irradiation, often combined with a small number of standard-of-care chemotherapeutic drugs, preferentially for advanced tumour patients. Only very recently, newer targeted therapies have entered the clinics, including Cetuximab, which targets the EGF receptor (EGFR), and several immune checkpoint inhibitors targeting the immune receptor PD-1 and its ligand PD-L1. HNSCC tumour tissues are characterized by a high degree of intra-tumour heterogeneity (ITH), and non-genetic alterations that may affect both non-transformed cells, such as cancer-associated fibroblasts (CAFs), and transformed carcinoma cells. This very high degree of heterogeneity likely contributes to acquired drug resistance, tumour dormancy, relapse, and distant or lymph node metastasis. ITH, in turn, is likely promoted by pronounced tumour cell plasticity, which manifests in highly dynamic and reversible phenomena such as of partial or hybrid forms of epithelial-to-mesenchymal transition (EMT), and enhanced tumour stemness. Stemness and tumour cell plasticity are strongly promoted by Notch signalling, which remains poorly understood especially in HNSCC. Here, we aim to elucidate how Notch signal may act both as a tumour suppressor and proto-oncogenic, probably during different stages of tumour cell initiation and progression. Notch signalling also interacts with numerous other signalling pathways, that may also have a decisive impact on tumour cell plasticity, acquired radio/chemoresistance, and metastatic progression of HNSCC. We outline the current stage of research related to Notch signalling, and how this pathway may be intricately interconnected with other, druggable targets and signalling mechanisms in HNSCC.

Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis.

The Notch signaling pathway is a critical player in embryogenesis but also plays various roles in tumorigenesis, with both tumor suppressor and oncogenic activities. Mutations, deletions, amplifications, or over-expression of Notch receptors, ligands, and a growing list of downstream Notch-activated genes have by now been described for most human cancer types. Yet, it often remains unclear what may be the functional impact of these changes for tumor biology, initiation, and progression, for cancer therapy, and for personalized medicine. Emerging data indicate that Notch signaling can also contribute to increased aggressive properties such as invasion, tumor heterogeneity, angiogenesis, or tumor cell dormancy within solid cancer tissues; especially in epithelial cancers, which are in the center of this review. Notch further supports the "stemness" of cancer cells and helps define the stem cell niche for their long-term survival, by integrating the interaction between cancer cells and the cells of the tumor microenvironment (TME). The complexity of Notch crosstalk with other signaling pathways and its roles in cell fate and trans-differentiation processes such as epithelial-to-mesenchymal transition (EMT) point to this pathway as a decisive player that may tip the balance between tumor suppression and promotion, differentiation and invasion. Here we not only review the literature, but also explore genomic databases with a specific focus on Notch signatures, and how they relate to different stages in tumor development. Altered Notch signaling hereby plays a key role for tumor cell survival and coping with a broad spectrum of vital issues, contributing to failed therapies, poor patient outcome, and loss of lives.

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products.

Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.