Colorectal cancer is a heterogeneous disease that manifests through diverse clinical scenarios. During many years, our knowledge about the variability of colorectal tumors was limited to the histopathological analysis from which generic classifications associated with different clinical expectations are derived. However, currently we are beginning to understand that under the intense pathological and clinical variability of these tumors there underlies strong genetic and biological heterogeneity. Thus, with the increasing available information of inter-tumor and intra-tumor heterogeneity, the classical pathological approach is being displaced in favor of novel molecular classifications. In the present article, we summarize the most relevant proposals of molecular classifications obtained from the analysis of colorectal tumors using powerful high throughput techniques and devices. We also discuss the role that cancer systems biology may play in the integration and interpretation of the high amount of data generated and the challenges to be addressed in the future development of precision oncology. In addition, we review the current state of implementation of these novel tools in the pathological laboratory and in clinical practice.

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression.

The use of one-step nucleic acid amplification (OSNA) allows for lymph node (LN) metastasis to be detected rapidly and accurately. We conducted a prospective single-centre clinical trial to evaluate OSNA assay in detecting LN metastasis of lung cancer. A total of 705 LNs from 160 patients with clinical stage IA to IVA lung cancer were included in this study. The LNs were divided and submitted to routine histological diagnosis and OSNA assay and the results were compared. We also examined keratin 19 expression of different histological types lung primary tumours. When the cut-off value was set to 250 copies/µl, the concordance rate between the two methods was 96.17% and the sensitivity 97.14%. Discordant results were observed in 27 LNs of 21 patients. Most of these discordant results were molecular micrometastasis expressing a very low number of copies with negative histology. Most thoracic tumours were positive for keratin 19. Our data show that the OSNA assay might be a useful and sensitive method to diagnose LN metastasis in lung cancer and could be applied to intraoperative decision-making in personalised lung cancer surgery based on LN status and a more accurate staging of patients.

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.

Tumor-derived extracellular vesicles (TD-EVs) have active roles as cancer hallmark enablers. EVs RNA of epithelial and stromal cells carry information that facilitates the communication processes that contribute to oncological progression, so the objective of this work was to validate by RT-PCR the presence of epithelial (KRT19; CEA) and stromal (COL1A2; COL11A1) markers in RNA of plasmatic EVs in healthy and diverse-malignancy patients for the development of a non-invasive cancer diagnosis system using liquid biopsy. Ten asymptomatic controls and 20 cancer patients were included in the study, and results showed that the isolated plasmatic EVs by scanning transmission electron microscopy (STEM) andBiomedical Research Institute A Coruña nanoparticle tracking analysis (NTA) contained most exosome structures with also a considerable percentage of microvesicles. No differences were found in concentration and size distribution between the two cohorts of patients, but significant gene expression in epithelial and mesenchymal markers between healthy donors and patients with active oncological disease was shown. Results of quantitative RT-PCR are solid and reliable for KRT19, COL1A2, and COL11A1, so the analysis of RNA extracted from TD-EVs could be a correct approach to develop a diagnostic tool in oncological processes.

Stage II colorectal cancer (CRC) recurrence remains a clinical problem. Some of these patients are true stage III CRC with a pN0 pathology stage. This large prospective multicentre cohort study aimed at evaluating the diagnostic ability of lymph node (LN) cytology smears to perform the pN stage and compare it with the conventional haematoxylin and eosin (H&E) pathology pN stage. Additionally, we used the One-Step Nucleic Acid Amplification (OSNA), a high-sensitive molecular method of LN staging. A total of 3936 fresh LNs from 217 CRC surgical specimens were examined by three methods, H&E, LN cytology smears, and OSNA. H&E detected 29% of patients with positive LNs, cytology smears 35%, and OSNA 33.2% (p < 0.0001). H&E and cytology concordantly classified 92.2% of tumours, and 88.5% between OSNA and H&E. Cytology had 96.8% sensitivity and 90.3% specificity to discriminate positive/negative patients compared to H&E (p = 0.004), and 87.3% sensitivity and 89% specificity when compared to OSNA (p = 0.56). Patients with positive LNs detected by any of the three methods had significantly worse disease-free and overall survival. We conclude that pN stage accuracy for detecting positive LNs is superior with LN cytological smears than with conventional H&E, which would enable a better pN stage and management of early-stage CRC patients.