Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer.

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

CD44v6, STn & O-GD2: promising tumor associated antigens paving the way for new targeted cancer therapies.

Targeted therapies are the state of the art in oncology today, and every year new Tumor-associated antigens (TAAs) are developed for preclinical research and clinical trials, but few of them really change the therapeutic scenario. Difficulties, either to find antigens that are solely expressed in tumors or the generation of good binders to these antigens, represent a major bottleneck. Specialized cellular mechanisms, such as differential splicing and glycosylation processes, are a good source of neo-antigen expression. Changes in these processes generate surface proteins that, instead of showing decreased or increased antigen expression driven by enhanced mRNA processing, are aberrant in nature and therefore more specific targets to elicit a precise anti-tumor therapy. Here, we present promising TAAs demonstrated to be potential targets for cancer monitoring, targeted therapy and the generation of new immunotherapy tools, such as recombinant antibodies and chimeric antigen receptor (CAR) T cell (CAR-T) or Chimeric Antigen Receptor-Engineered Natural Killer (CAR-NK) for specific tumor killing, in a wide variety of tumor types. Specifically, this review is a detailed update on TAAs CD44v6, STn and O-GD2, describing their origin as well as their current and potential use as disease biomarker and therapeutic target in a diversity of tumor types.

PARP inhibition promotes endothelial-like traits in melanoma cells and modulates pericyte coverage dynamics during vasculogenic mimicry.

Vasculogenic mimicry (VM) describes the ability of highly aggressive tumor cells to develop pseudovascular structures without the participation of endothelial cells. PARP1 is implicated in the activation of hypoxia-inducible factors, which are crucial in tumor neovascularization. We have explored the role of hypoxia and PARP inhibition in VM. In uveal melanoma xenografts, the PARP inhibitor olaparib improved in vivo pericyte coverage specifically of VM channels. This was concomitant with reduced metastasis in olaparib-treated VM+ tumors. PARP inhibition and hypoxia modulated melanoma tube formation in vitro, inducing a more sparse and regular tubular architecture. Whole-transcriptome profiling revealed that olaparib treatment under hypoxic conditions modulated the expression of genes implicated in vasculogenesis during tube formation, enhancing the endothelial-like phenotype of VM+ uveal melanoma cells. PARP inhibition, especially during hypoxia, upregulated PDGFß, which is essential for pericyte recruitment. Our study indicates that PARP inhibitors may enhance the endothelial characteristics of VM+ cells, modulate pericyte coverage, and reduce metastatic spread in VM+ melanoma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Pten and p53 Loss in the Mouse Lung Causes Adenocarcinoma and Sarcomatoid Carcinoma.

Lung cancer remains the leading cause of cancer deaths worldwide. Among the Non-Small Cell Carcinoma (NSCLC) category, Adenocarcinoma (ADC) represents the most common type, with different reported driver mutations, a bunch of models described and therapeutic options. Meanwhile, Pulmonary Sarcomatoid Carcinoma (PSC) is one of the rarest, with very poor outcomes, scarce availability of patient material, no effective therapies and no models available for preclinical research. Here, we describe that the combined deletion of Pten and Trp53 in the lungs of adult conditional mice leads to the development of both ADC and PSC irrespective of the lung targeted cell type after naphthalene induced airway epithelial regeneration. Although this model shows long latency periods and incomplete penetrance for tumor development, it is the first PSC mouse model reported so far, and sheds light on the relationships between ADC and PSC and their cells of origin. Moreover, human ADC show strong transcriptomic similarities to the mouse PSC, providing a link between both tumor types and the human ADC.

Analysis of Exosomal Cargo Provides Accurate Clinical, Histologic and Mutational Information in Non-Small Cell Lung Cancer.

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes' cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease.

G9a inhibition by CM-272: Developing a novel anti-tumoral strategy for castration-resistant prostate cancer using 2D and 3D in vitro models.

Castration-resistant prostate cancer (CRPC) is an incurable form of prostate cancer (PCa), with DNMT1 and G9a being reported as overexpressed, rendering them highly attractive targets for precision medicine. CM-272 is a dual inhibitor of both methyltransferases' activity. Herein, we assessed the response of different PCa cell lines to CM-272, in both 2D and 3D models, and explored the molecular mechanisms underlying CM-272 inhibitory effects. CRPC tissues displayed significantly higher DNMT1, G9a and H3K9me2 expression than localized PCa. In vitro, CM-272 caused a significant decrease in PCa cell viability and proliferation alongside with increased apoptotic levels. We disclose that, under the evaluated dose, CM-272 led to G9a activity inhibition, while not significantly affecting DNMT1 activity. Upon G9a knockdown, DU145 and PC3 showed decreased cell viability. Remarkably, DU145 cells treated with CM-272 or with G9a knockdown displayed no differences in viability, suggesting a SET-dependent mechanism. Contrarily, PC3 cell viability impact was higher in G9a knockdown, compared with CM-272 treatment, suggesting an additional G9a function. Moreover, DU145 cells overexpressing catalytically functional G9a disclosed higher resistance to CM-272 treatment, reinforcing that the drug mechanism of action is dependent on G9a catalytic function. Importantly, we successfully assembled spheroids from several prostate cell lines. Our results showed that CM-272 retained its anti-tumoral effects in 3D PCa models, leading to a clear reduction in cancer cell survival. We concluded that inhibition of G9a methyltransferase activity by CM-272 has anti-tumor effect in PCa cells, holding therapeutic potential against CRPC.

IKKα Induces Epithelial-Mesenchymal Changes in Mouse Skin Carcinoma Cells That Can Be Partially Reversed by Apigenin.

NMSC (non-melanoma skin cancer) is a common tumor in the Caucasian population, accounting for 90% of skin cancers. Among them, squamous cell carcinomas (SCCs) can metastasize and, due to its high incidence, constitute a severe health problem. It has been suggested that cutaneous SCCs with more risk to metastasize express high levels of nuclear IKKα. However, the molecular mechanisms that lead to this enhanced aggressiveness are largely unknown. To understand in depth the influence of nuclear IKKα in skin SCC progression, we have generated murine PDVC57 skin carcinoma cells expressing exogenous IKKα either in the nucleus or in the cytoplasm to further distinguish the tumor properties of IKKα in both localizations. Our results show that IKKα promotes changes in both subcellular compartments, resembling EMT (epithelial-mesenchymal transition), which are more pronounced when IKKα is in the nucleus of these tumor cells. These EMT-related changes include a shift toward a migratory phenotype and induction of the expression of proteins involved in cell matrix degradation, cell survival and resistance to apoptosis. Additionally, we have found that apigenin, a flavonoid with anti-cancer properties, inhibits the expression of IKKα and attenuates most of the pro-tumoral EMT changes induced by IKKα in mouse tumor keratinocytes. Nevertheless, we have found that apigenin only inhibits the expression of the IKKα protein when it is localized in the cytoplasm.

Toward Tumor Fight and Tumor Microenvironment Remodeling: PBA Induces Cell Cycle Arrest and Reduces Tumor Hybrid Cells' Pluripotency in Bladder Cancer.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1ß. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b+EPCAM+) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers (SOX2, NANOG, miR-302) as compared with non-fused (CD11b-EPCAM+) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10. Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

Genomic Landscape of Vinflunine Response in Metastatic Urothelial Cancer.

BACKGROUND AND AIMS: Metastatic urothelial carcinoma (mUC) remains an incurable disease with limited treatment options after platinum-based chemotherapy and immune checkpoint blockade (ICB). Vinflunine has shown a modest increase in overall survival and remains a therapeutic option for chemo- and immunotherapy refractory tumours. However, biomarkers that could identify responding patients to vinflunine and possible alternative therapies after failure to treatment are still missing. In this study, we aimed to identify potential genomic biomarkers of vinflunine response in mUC patient samples and potential management alternatives. METHODS: Formalin-fixed paraffin-embedded samples of mUC patients (n = 23) from three university hospitals in Spain were used for genomic targeted-sequencing and transcriptome (using the Immune Profile panel by NanoString) analyses. Patients who received vinflunine after platinum-based chemotherapy failure were classified in non-responders (NR: progressive disease ≤ 3 months; n= 11) or responders (R: response ≥ 6 months; n = 12). RESULTS: Genomic characterization revealed that the most common alteration, TP53 mutations, had comparable frequency in R (6/12; 50%) and NR (4/11; 36%). Non-synonymous mutations in KTM2C (4/12; 33.3%), PIK3CA (3/12; 25%) and ARID2 (3/12; 25%) were predominantly associated with response. No significant difference was observed in tumour mutational burden (TMB) between R and NR patients. The NR tumours showed increased expression of diverse immune-related genes and pathways, including various interferon gamma-related genes. We also identified increased MAGEA4 expression as a potential biomarker of non-responding tumours to vinflunine treatment. CONCLUSIONS: Our data may help to identify potential genomic biomarkers of response to vinflunine. Moreover, tumours refractory to vinflunine showed immune signatures potentially associated with response to ICB. Extensive validation studies, including longitudinal series, are needed to corroborate these findings.

CYLD Inhibits the Development of Skin Squamous Cell Tumors in Immunocompetent Mice.

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.

Functional Specificity of the Members of the Sos Family of Ras-GEF Activators: Novel Role of Sos2 in Control of Epidermal Stem Cell Homeostasis.

Prior reports showed the critical requirement of Sos1 for epithelial carcinogenesis, but the specific functionalities of the homologous Sos1 and Sos2 GEFs in skin homeostasis and tumorigenesis remain unclear. Here, we characterize specific mechanistic roles played by Sos1 or Sos2 in primary mouse keratinocytes (a prevalent skin cell lineage) under different experimental conditions. Functional analyses of actively growing primary keratinocytes of relevant genotypes-WT, Sos1-KO, Sos2-KO, and Sos1/2-DKO-revealed a prevalent role of Sos1 regarding transcriptional regulation and control of RAS activation and mechanistic overlapping of Sos1 and Sos2 regarding cell proliferation and survival, with dominant contribution of Sos1 to the RAS-ERK axis and Sos2 to the RAS-PI3K/AKT axis. Sos1/2-DKO keratinocytes could not grow under 3D culture conditions, but single Sos1-KO and Sos2-KO keratinocytes were able to form pseudoepidermis structures that showed disorganized layer structure, reduced proliferation, and increased apoptosis in comparison with WT 3D cultures. Remarkably, analysis of the skin of both newborn and adult Sos2-KO mice uncovered a significant reduction of the population of stem cells located in hair follicles. These data confirm that Sos1 and Sos2 play specific, cell-autonomous functions in primary keratinocytes and reveal a novel, essential role of Sos2 in control of epidermal stem cell homeostasis.

Cell Therapies in Bladder Cancer Management.

Currently, bladder cancer (BC) represents a challenging problem in the field of Oncology. The high incidence, prevalence, and progression of BC have led to the exploration of new avenues in its management, in particular in advanced metastatic stages. The recent inclusion of immune checkpoint blockade inhibitors as a therapeutic option for BC represents an unprecedented advance in BC management. However, although some patients show durable responses, the fraction of patients showing benefit is still limited. Notwithstanding, cell-based therapies, initially developed for the management of hematological cancers by infusing immune or trained immune cells or after the engineering of chimeric antigen receptor (CAR) expressing cells, are promising tools to control, or even cure, solid tumors. In this review, we summarize recent cell-based immunotherapy studies, with a special focus on BC.

Tackling tumor microenvironment through epigenetic tools to improve cancer immunotherapy.

BACKGROUND: Epigenetic alterations are known contributors to cancer development and aggressiveness. Additional to alterations in cancer cells, aberrant epigenetic marks are present in cells of the tumor microenvironment, including lymphocytes and tumor-associated macrophages, which are often overlooked but known to be a contributing factor to a favorable environment for tumor growth. Therefore, the main aim of this review is to give an overview of the epigenetic alterations affecting immune cells in the tumor microenvironment to provoke an immunosuppressive function and contribute to cancer development. Moreover, immunotherapy is briefly discussed in the context of epigenetics, describing both its combination with epigenetic drugs and the need for epigenetic biomarkers to predict response to immune checkpoint blockage. MAIN BODY: Combining both topics, epigenetic machinery plays a central role in generating an immunosuppressive environment for cancer growth, which creates a barrier for immunotherapy to be successful. Furthermore, epigenetic-directed compounds may not only affect cancer cells but also immune cells in the tumor microenvironment, which could be beneficial for the clinical response to immunotherapy. CONCLUSION: Thus, modulating epigenetics in combination with immunotherapy might be a promising therapeutic option to improve the success of this therapy. Further studies are necessary to (1) understand in depth the impact of the epigenetic machinery in the tumor microenvironment; (2) how the epigenetic machinery can be modulated according to tumor type to increase response to immunotherapy and (3) find reliable biomarkers for a better selection of patients eligible to immunotherapy.

Phenotypic Analysis of Urothelial Exfoliated Cells in Bladder Cancer via Microfluidic Immunoassays: Sialyl-Tn as a Novel Biomarker in Liquid Biopsies.

Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes. in situ immunoassays demonstrated significantly higher number of Sialyl-Tn-positive cells in fresh and frozen voided urine from bladder cancer patients, compared to healthy individuals. Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies.

VAV2 signaling promotes regenerative proliferation in both cutaneous and head and neck squamous cell carcinoma.

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.

DNA Methylation as a Therapeutic Target for Bladder Cancer.

Bladder cancer (BC) is the tenth most frequent cancer worldwide and is associated with high mortality when diagnosed in its most aggressive form, which is not reverted by the current treatment options. Thus, the development of new therapeutic strategies, either alternative or complementary to the current ones, is of major importance. The disruption of normal epigenetic mechanisms, namely, DNA methylation, is a known early event in cancer development. Consequently, DNA methyltransferase (DNMT) inhibitors constitute a promising therapeutic target for the treatment of BC. Although these inhibitors, mainly nucleoside analogues such as 5-azacytidine (5-aza) and decitabine (DAC), cause re-expression of tumor suppressor genes, inhibition of tumor cell growth, and increased apoptosis in BC experimental models and clinical trials, they also show important drawbacks that prevent their use as a valuable option for the treatment of BC. However, their combination with chemotherapy and/or immune-checkpoint inhibitors could aid in their implementation in the clinical practice. Here, we provide a comprehensive review of the studies exploring the effects of DNA methylation inhibition using DNMTs inhibitors in BC, from in vitro and in vivo studies to clinical trials.

Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer.

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

Competitive Repopulation Assay of Long-Term Epidermal Stem Cell Regeneration Potential.

Epidermal stem cells are responsible for normal tissue homeostasis and contribute to tissue regeneration during injury. Several assays measuring stem cell frequency and function can be used to assess epidermal stem cell potential. However, the ultimate assay that accounts for stemness is the capacity to sustain in vivo long-term tissue regeneration and maintenance. We can use this type of analysis to interrogate whether a specific genetic alteration (e.g., activation or inactivation of any gene thought to be involved in stem cell quiescence or proliferation) confers increased or decreased stem cell potential.

G-protein-coupled receptor kinase 2 safeguards epithelial phenotype in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.