Biology and Therapeutic Targets of Colorectal Serrated Adenocarcinoma; Clues for a Histologically Based Treatment against an Aggressive Tumor.

Serrated adenocarcinoma (SAC) is a tumor recognized by the WHO as a histological subtype accounting for around 9% of colorectal carcinomas. Compared to conventional carcinomas, SACs are characterized by a worse prognosis, weak development of the immune response, an active invasive front and a frequent resistance to targeted therapy due to a high occurrence of KRAS or BRAF mutation. Nonetheless, several high-throughput studies have recently been carried out unveiling the biology of this cancer and identifying potential molecular targets, favoring a future histologically based treatment. This review revises the current evidence, aiming to propose potential molecular targets and specific treatments for this aggressive tumor.

New role of the antidepressant imipramine as a Fascin1 inhibitor in colorectal cancer cells.

Serrated adenocarcinoma (SAC) is more invasive, has worse outcomes than conventional colorectal carcinoma (CRC), and is characterized by frequent resistance to anti-epidermal growth factor receptor (EGFR) and overexpression of fascin1, a key protein in actin bundling that plays a causative role in tumor invasion and is overexpressed in different cancer types with poor prognosis. In silico screening of 9591 compounds, including 2037 approved by the Food and Drug Administration (FDA), was performed, and selected compounds were analyzed for their fascin1 binding affinity by differential scanning fluorescence. The results were compared with migrastatin as a typical fascin1 inhibitor. In silico screening and differential scanning fluorescence yielded the FDA-approved antidepressant imipramine as the most evident potential fascin1 blocker. Biophysical and different in vitro actin-bundling assays confirm this activity. Subsequent assays investigating lamellipodia formation and migration and invasion of colorectal cancer cells in vitro using 3D human tissue demonstrated anti-fascin1 and anti-invasive activities of imipramine. Furthermore, expression profiling suggests the activity of imipramine on the actin cytoskeleton. Moreover, in vivo studies using a zebrafish invasion model showed that imipramine is tolerated, its anti-invasive and antimetastatic activities are dose-dependent, and it is associated with both constitutive and induced fascin1 expression. This is the first study that demonstrates an antitumoral role of imipramine as a fascin1 inhibitor and constitutes a foundation for a molecular targeted therapy for SAC and other fascin1-overexpressing tumors.

Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression.

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.

Opposing roles of PIK3CA gene alterations to EZH2 signaling in non-muscle invasive bladder cancer.

The high rates of tumor recurrence and progression represent a major clinical problem in non-muscle invasive bladder cancer. Previous data showed that EZH2-dependent signaling mediates these processes, whereas the frequent alterations of PIK3CA gene (copy gains and mutations) are predictive of reduced recurrence. Here we show, using clinical samples and bladder cancer cell lines, a functional interaction between EZH2- and PIK3CA-dependent signaling pathways. PIK3CA alterations mediated, on the one hand, the increased expression of two miRNAs, miR-101 and miR-138, which posttranscriptionally downregulate EZH2 expression. On the other hand, PIK3CA alterations facilitate the activation of Akt which phosphorylates EZH2 on Ser21, precluding the trimethylation of histone H3 in K27. Remarkably the increased expression of miR101 or miR138 and the expression of Ser21-phosphorylated EZH2 are good prognostic factors regarding non-muscle invasive bladder cancer recurrence and progression. Collectively, this study provides molecular evidences indicating that the gene expression rewiring occurring in primary bladder tumors, associated with increased EZH2 expression and activity and mediating the increased recurrence and progression risk, are prevented by PIK3CA-dependent signaling. This molecular process may have deep implications in the management of bladder cancer patients and in the design of novel molecularly targeted therapeutic approaches.

EZH2 in Bladder Cancer, a Promising Therapeutic Target.

Bladder Cancer (BC) represents a current clinical and social challenge. The recent studies aimed to describe the genomic landscape of BC have underscored the relevance of epigenetic alterations in the pathogenesis of these tumors. Among the epigenetic alterations, histone modifications occupied a central role not only in cancer, but also in normal organism homeostasis and development. EZH2 (Enhancer of Zeste Homolog 2) belongs to the Polycomb repressive complex 2 as its catalytic subunit, which through the trimethylation of H3 (Histone 3) on K27 (Lysine 27), produces gene silencing. EZH2 is frequently overexpressed in multiple tumor types, including BC, and plays multiple roles besides the well-recognized histone mark generation. In this review, we summarize the present knowledge on the oncogenic roles of EZH2 and its potential use as a therapeutic target, with special emphasis on BC pathogenesis and management.

A Polycomb-mir200 loop regulates clinical outcome in bladder cancer.

Bladder cancer (BC) is a highly prevalent disease, ranking fifth in the most common cancers worldwide. Various miRNAs have recently emerged as potential prognostic biomarkers in cancer. The miR-200 family, which repressed the epithelial-to-mesenchymal transition (EMT), is repressed in multiple advanced cancers. However, its expression and function in BC is still poorly understood. Here we show that miR-200 family displays increased expression, probably due to the activation of specific oncogenic signaling pathways, and reduced promoter methylation, in BC compared to normal bladder samples. Furthermore, we show that the expression of these miRNAs is decreased in high grade and stage tumors, and the down-regulation is associated with patient's poor clinical outcome. Our data indicate that the miR-200 family plays distinct roles in Non-Muscle (NMIBC) and Muscle-Invasive BC (MIBC). In MIBC, miR-200 expression post transcriptionally regulates EMT-promoting transcription factors ZEB1 and ZEB2, whereas suppresses BMI1 expression in NMIBC. Interestingly, we show that increased EZH2 and/or BMI1 expression repress the expression of miR-200 family members. Collectively, these findings support a model of BC progression through a coordinated action between the Polycomb Repression Complex (PRC) members repressing the miR-200 expression, which ultimately favors invasive BC development. Since pharmacological inhibition of EZH2 in BC cell lines lead to increased miR-200 expression, our findings may support new therapeutic strategies for BC clinical management.

Analysis of the Polycomb-related lncRNAs HOTAIR and ANRIL in bladder cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been claimed as key molecular players in gene expression regulation, being involved in diverse epigenetic processes. They are aberrantly expressed in various tumors, but their exact role in bladder cancer is still obscure. We have recently found a major role of the Polycomb repression complex in recurrence of non-muscle-invasive bladder cancer. Here, we report the xpression of Polycomb-related lncRNAs:antisense noncoding RNA in the INK4 locus (ANRIL) and HOX antisense intergenic RNA (HOTAIR) in these tumors. FINDINGS: We studied a dataset of non-invasive bladder cancer samples by quantitative reverse transcription PCR (RT-qPCR) and analyzed also invasive bladder cancer samples using TCGA data. Our results showed that, while ANRIL seemed not to have a determining role, an increased HOTAIR expression appeared in recurrent and high-graded tumors associated with poor prognosis. In addition, through genome-wide transcriptome analyses, we observed that HOTAIR-EZH2-complex-regulated genes can efficiently discriminate between non-tumoral, recurrent, and non-recurrent bladder cancer samples. We also observed a significant correlation between EZH2 and HOTAIR expression levels. Using overexpression, knockdown, and pharmacological approaches in bladder cancer cell lines, we also observed that EZH2 regulates HOTAIR expression. CONCLUSIONS: Our findings indicate that HOTAIR expression has prognostic value for bladder cancer progression, recurrence, and survival and suggest that HOTAIR plays active roles in modulating the cancer epigenome, becoming an interesting candidate as a target for cancer diagnosis and therapy. The observed HOTAIR regulation by EZH2 and the possibility of modulating EZH2 activity with specific inhibitors open new possible paths to be explored in bladder cancer therapy.

The downregulation of ΔNp63 in p53-deficient mouse epidermal tumors favors metastatic behavior.

The TP63 gene codes for two major isoform types, TAp63 and ΔNp63, with probable opposite roles in tumorigenesis. The ΔNp63α protein is frequently amplified and overexpressed in different epithelial tumors. Accordingly, it has been considered a potential oncogene. Nonetheless, a possible metastatic suppressor activity has also been suggested based on the experimental observation that its expression is reduced or even absent in advanced invasive tumors. Such metastatic suppressor activities are often related to tumors bearing point mutated TP53 gene. However, its potential roles in TP53-deficient tumors are poorly characterized. Here we show that in spontaneous tumors, induced by the epidermal-specific Trp53 ablation, the reduction of ΔNp63 expression is an early event, whereas it is re-expressed in the lung metastatic lesions. Using knock down and ectopic expression approaches, we show that ΔNp63 expression opposes the epithelial-mesenchymal transition and reduces the metastatic potential of the cells. This process occurs through the modulation of ΔNp63-dependent downstream targets (including transcription factors and microRNAs) likely to play metastatic roles. Further, ΔNp63 also favors the expression of factors involved in iPS reprogramming, thus suggesting that it can also modulate specific stem cell traits in mouse epidermal tumor cells. Overall, our data assign antimetastatic roles to ΔNp63 in the context of p53 deficiency and epidermis.