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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, β-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.

Wnt-11 promotes cancer cell migration and invasion independently of β-catenin but the receptors involved remain unknown. Here, we provide evidence that FZD 8 is a major Wnt-11 receptor in prostate cancer that integrates Wnt-11 and TGF-β signals to promote EMT. FZD8 mRNA is upregulated in multiple prostate cancer datasets and in metastatic cancer cell lines in vitro and in vivo. Analysis of patient samples reveals increased levels of FZD 8 in cancer, correlating with Wnt-11. FZD 8 co-localizes and co-immunoprecipitates with Wnt-11 and potentiates Wnt-11 activation of ATF2-dependent transcription. FZD8 silencing reduces prostate cancer cell migration, invasion, three-dimensional (3D) organotypic cell growth, expression of EMT-related genes, and TGF-β/Smad-dependent signaling. Mechanistically, FZD 8 forms a TGF-β-regulated complex with TGF-β receptors that is mediated by the extracellular domains of FZD 8 and TGFBR1. Targeting FZD 8 may therefore inhibit aberrant activation of both Wnt and TGF-β signals in prostate cancer. Wnt11 has been shown to play a role in invasion and metastasis of prostate cancer. Here the authors show that in prostate cancer cells Wnt11 signals through the Fzd8 receptor and report an interaction between Fzd8 and TGF-β receptors regulating the transcription of a subset of TGF-beta genes.

Prostate epithelial cells from both normal and cancer tissues, grown in three-dimensional (3D) culture as spheroids, represent promising in vitro models for the study of normal and cancer-relevant patterns of epithelial differentiation. We have developed the most comprehensive panel of miniaturized prostate cell culture models in 3D to date (n = 29), including many non-transformed and most currently available classic prostate cancer (PrCa) cell lines. The purpose of this study was to analyze morphogenetic properties of PrCa models in 3D, to compare phenotypes, gene expression and metabolism between 2D and 3D cultures, and to evaluate their relevance for pre-clinical drug discovery, disease modeling and basic research. Primary and non-transformed prostate epithelial cells, but also several PrCa lines, formed well-differentiated round spheroids. These showed strong cell-cell contacts, epithelial polarization, a hollow lumen and were covered by a complete basal lamina (BL). Most PrCa lines, however, formed large, poorly differentiated spheroids, or aggressively invading structures. In PC-3 and PC-3M cells, well-differentiated spheroids formed, which were then spontaneously transformed into highly invasive cells. These cell lines may have previously undergone an epithelial-to-mesenchymal transition (EMT), which is temporarily suppressed in favor of epithelial maturation by signals from the extracellular matrix (ECM). The induction of lipid and steroid metabolism, epigenetic reprogramming, and ECM remodeling represents a general adaptation to 3D culture, regardless of transformation and phenotype. In contrast, PI3-Kinase, AKT, STAT/interferon and integrin signaling pathways were particularly activated in invasive cells. Specific small molecule inhibitors targeted against PI3-Kinase blocked invasive cell growth more effectively in 3D than in 2D monolayer culture, or the growth of normal cells. Our panel of cell models, spanning a wide spectrum of phenotypic plasticity, supports the investigation of different modes of cell migration and tumor morphologies, and will be useful for predictive testing of anti-cancer and anti-metastatic compounds.

Head and neck cancer (HNC) is highly heterogeneous and difficult to treat, underscoring the need for rapid, patient-specific models. Standard three-dimensional (3D) cultures often lose stromal partners that influence therapy response. We developed a patient-derived system maintaining tumor cells, cancer-associated fibroblasts (CAFs), and cells undergoing partial epithelial–mesenchymal transition (pEMT) for drug sensitivity testing. Biopsies from four HNC patients were enzymatically dissociated. CAFs were directly cultured, and their conditioned medium (CAF-CM) was collected. Cryopreserved primary tumor cell suspensions were later revived, screened in five different growth media under 2D conditions, and the most heterogeneous cultures were re-embedded in 3D hydrogels with varied gel mixtures, media, and seeding geometries. Tumoroid morphology was quantified using a perimeter-based complexity index. Viability after treatment with cisplatin or Notch modulators (RIN-1, recombination signal-binding protein for immunoglobulin κ J region (RBPJ) inhibitor; FLI-06, inhibitor) was assessed by live imaging and the water-soluble tetrazolium-8 (WST-8) assay. Endothelial Cell Growth Medium 2 (ECM-2) medium alone produced compact CAF-free spheroids, whereas ECM-2 supplemented with CAF-CM generated invasive aggregates that deposited endogenous matrix. Matrigel with this medium and single-point seeding gave the highest complexity scores. Two of the three patient tumoroids were cisplatin-sensitive, and all showed significant growth inhibition with the FLI-06 Notch inhibitor, while the RBPJ inhibitor RIN-1 induced minimal change. The optimized scaffold retains tumor–stroma crosstalk and provides patient-specific drug response data within days after operation, supporting personalized treatment selection in HNC.

Sirtuin 7 (SIRT7), a member of the sirtuin family of NAD+-dependent deacetylases, plays a vital role in cancer, exhibiting context-dependent functions across various malignancies. Our study investigates the role of SIRT7 depletion in head and neck squamous cell carcinoma (HNSCC) progression. In vitro and 3D organotypic models demonstrated that SIRT7 knock-out attenuates cancer cell viability, proliferation, and motility as well as induces downregulation of migration- and epithelial-mesenchymal transition (EMT)-related gene expression. Moreover, the SIRT7 loss results in slower organoid formation and less invasive organoid morphology, validated by vimentin downregulation. The SIRT7 loss potentiates S-phase arrest in cell cycle progression after 5-FU treatment and elevates the ratio of dead cells. Additionally, SIRT7 deletion reduces the expression of G1 phase-associated proteins, Cyclin D and CDK4. Altogether, our study highlights SIRT7 as a promising therapeutic target in HNSCC, enhancing the effectiveness of treatment modalities such as combinational treatment.

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.

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.

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.

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases.