EPMA position paper in cancer:current overview and future perspectives

At present, a radical shift in cancer treatment is occurring in terms of predictive, preventive, and personalized medicine (PPPM). Individual patients will participate in more aspects of their healthcare. During the development of PPPM, many rapid, specific, and sensitive new methods for earlier detection of cancer will result in more efficient management of the patient and hence a better quality of life. Coordination of the various activities among different healthcare professionals in primary, secondary, and tertiary care requires well-defined competencies, implementation of training and educational programs, sharing of data, and harmonized guidelines. In this position paper, the current knowledge to understand cancer predisposition and risk factors, the cellular biology of cancer, predictive markers and treatment outcome, the improvement in technologies in screening and diagnosis, and provision of better drug development solutions are discussed in the context of a better implementation of personalized medicine. Recognition of the major risk factors for cancer initiation is the key for preventive strategies (EPMA J. 4(1):6, 2013). Of interest, cancer predisposing syndromes in particular the monogenic subtypes that lead to cancer progression are well defined and one should focus on implementation strategies to identify individuals at risk to allow preventive measures and early screening/diagnosis. Implementation of such measures is disturbed by improper use of the data, with breach of data protection as one of the risks to be heavily controlled. Population screening requires in depth cost-benefit analysis to justify healthcare costs, and the parameters screened should provide information that allow an actionable and deliverable solution, for better healthcare provision

HeLa cell line, a model to study the role of cofactor of BRCA1 in cervical cancer

Being one of the four subunits that makes up the Negative Elongation Factor Complex (NELF), Cofactor of BRCA1 (COBRA1); also known as NELF-B, is able to regulate a number of genes involved in cellular proliferation, metabolism, cell cycle progression and DNA repair. In addition, COBRA1 was shown to interact with other transcription factors such as BRCA1, AP-1 complex and several nuclear receptors. Despite the evidences that suggest COBRA1 as a potential player involved in the progression of a number of cancers, its role in cervical cancer has not been previously investigated. To date, it has been studied in breast, upper gastrointestinal and liver cancers. The main objective of our study was to investigate the potential involvement of COBRA1 in cervical cancer progression. We first did in-silico analysis of the expression patterns of COBRA1 in cervical cancer tissues relative to normal cervical tissues using the publicly available Oncomine Cancer Microarray Database. Search results revealed a significant upregulation of COBRA1 in two mRNA microarray datasets. RNA interference technique was then used to knockdown COBRA1 expression in cervical cancer cell line, HeLa. Once a successful siRNA mediated silencing at the RNA and protein levels of COBRA1 was established and confirmed through semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot, we investigated its consequences on proliferation, migration and survival of HeLa cells. Interestingly, COBRA1 depletion resulted in a significant increase in the mRNA expression of Trefoil Factor 1 (TFF1) accompanied by a subsequent decrease in the β-catenin mRNA levels. These findings suggests an effect for COBRA1 on the Wnt/β-catenin signalling pathway, which could be mediated through TFF1. In addition, COBRA1 silencing resulted in significant decrease in the expression of survivin 2B and survivin DeltaEX3 isoforms while the observed decrease in survivin wild type form was found to be statistically insignificant. Survivin is known to play a major role in cancer cells proliferation and survival. Yet, the finding that the noted decrease in β-catenin and survivin expression was not reflected on the proliferation and migration abilities of HeLa is not conclusive and requires further investigations. Taken together, these findings could help as an initial step in identifying the role of COBRA1 in cervical cancer tumorigenesis

Towards an Understanding of the Evolution of the HPV E6 PDZ Binding Motif

The high-risk human alpha papillomaviruses, associated with the development of cervical cancer differ in the carboxyl-terminal sequence of the E6 oncogenic protein, where a PDZ-binding motif (PBM) is located. The PBM binds to PDZ-containing proteins of the cell, and variation in the PBM sequence is likely to affect the PDZ protein selectivity of E6, and the phospho-acceptor site embedded within the PBM. Using the E6 proteins from HPV types with diverse phylogenetic and risk classifications, we found that differences in their PBMs are reflected in their substrate selection. Moreover, we show that the ability of E6 proteins to target PDZ proteins has co-evolved with the phospho-regulation of the PBMs. Previously identified residues known to reduce both HPV-18 phosphorylation and its functional flexibility in substrate selection, were mutated in the core and upstream regions of HPV-66 and HPV-40 E6 PBMs to generate phospho-acceptor sites for CHK1, PKA and AKT kinases. The patterns of binding to PDZ proteins by these mutants were then evaluated. I found that the last residue of HPV-18 E6 (Valine) is critical for its recognition of MAGI-1 and when the last residue of HPV-40 E6 (Cysteine) is mutated to Valine, it increases HPV-40 E6's PBM-dependent binding to MAGI-1 in pull-down assays. This was also confirmed with Dlg1. As expected, the reverse (Valine to Cysteine) substitution in the HPV-18 E6 PBM also destroys its binding to Scribble, but the Cysteine to Valine substitution failed to confer Scribble binding on HPV-40, suggesting that additional critical residues are required for Scribble recognition. Additionally, we evaluated the phosphorylation of the HPV-66 E6 protein by the three kinases, introducing arginine residues on the p-4 and p-5 positions of the PBM. The wild-type HPV-66 E6 protein is not phosphorylated, as well as the p-5 mutant. On the contrary, mutations on p-4 and p-4/p-5 positions have increased phosphorylation by PKA, slightly by CHK1 but not by AKT. Mutations on p-5 position resulted in a mild increase in recognition of MAGI-1 whilst surprisingly mutations on p-4 which confers phosphorylation, does not. The arginine residue substitutions resulted in a mild increase of the ability of the HPV-66 E6 PBM to bind Dlg1, and failed to confer interaction with Scribble; again, suggesting the involvement of additional residues. Moreover, we used HPV-16 E6 protein to further evaluate if residues upstream of the PBM are important for binding PDZ proteins and whether upstream phosphor acceptor sites might regulate PDZ recognition. We evaluated a threonine residue on p-6 position and found that this position is phosphorylated by CK1 kinase with lower efficiency. Furthermore, the Aspartate substitution mildly increased binding to TIP-1 and SNTB2 PDZ proteins, and conversely, the Alanine substitution significantly decreased binding. Furthermore, we observed that a triplet of serine residues on positions p-8, p-9 and p-13 upstream of the PBM can be multiply phosphorylated by PKC and CK1 kinases. The multiple Aspartate substitution mutant showed higher retention of E6 in the cytoplasm, and the Alanine substitution mutant higher retention in the nucleus, indicating that phosphorylation of these residues might modulate E6 localisation. Taken together these studies demonstrate a unique linkage between evolution of the phospho-acceptor site embedded in the PBM and functional flexibility, together with further regulation provided by potentially unique phospho-acceptor sites only found in HPV-16 E6. Finally, we evaluated the capacity of the low-risk HPV-11, a less potent version of the HR types, but a common cause of benign tumours in the anogenital region and the upper respiratory tract, rarely associated with cancer, to degrade p53. Although, 11E6 can bind E6AP and p53, it binds the carboxy-terminal site of p53, and not the core domain that is necessary for degradation induced by the E6 of high-risk types. Regardless, a recent report has shown that HPV-11 E6 degrades p53 under high-confluency conditions, suggesting a conserved and common mechanism between high- and low-risk types. We showed in Part 3, that HPV-11 E6 degrades p53 after treatment with etoposide, a DNA damaging agent. It has been shown that Etoposide induces the phosphorylation of p53 on serine 15, an important and nucleating event in p53 activation through post-translational modifications. Furthermore, 11E6 induces the degradation of the p53 phospho-mimic mutant S15D, which mimics the activated form of p53 and which is not susceptible to Mdm2-induced degradation. Moreover, we confirmed that 11E6 co-localizes with p53 after the treatment with Etoposide, and with the p53 S15D phospho-mimic mutant. Interestingly, we observed that 11E6 influences the cellular distribution of wild type p53 to the cytoplasm, when transiently co-transfected together, as with the phospho-mimic mutant. We hypothesized that the common ancestor of high- and low-risk types might have been able to degrade specific forms of p53, and that eventually, the high-risk types developed the ability to degrade all forms of p53, contributing to their oncogenic potential. Whether 11E6’s specific ability to induce degradation of Ser15 phospho-p53 is related to HPV-11’s successful replication and maintenance within the cell needs investigating

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.Siirretty Doriast

Repurposing Antibacterial AM404 as a Potential Anticancer Drug for Targeting Colorectal Cancer Stem-Like Cells

Tumour-promoting inflammation is involved in colorectal cancer (CRC) development and therapeutic resistance. However, the antibiotics and antibacterial drugs and signalling that regulate the potency of anticancer treatment upon forced differentiation of cancer stem-like cell (CSC) are not fully defined yet. We screened an NIH-clinical collection of the small-molecule compound library of antibacterial/anti-inflammatory agents that identified potential candidate drugs targeting CRC-SC for differentiation. Selected compounds were validated in both in vitro organoids and ex vivo colon explant models for their differentiation induction, impediment on neoplastic cell growth, and to elucidate the mechanism of their anticancer activity. We initially focused on AM404, an anandamide uptake inhibitor. AM404 is a metabolite of acetaminophen with antibacterial activity, which showed high potential in preventing CRC-SC features, such as stemness/de-differentiation, migration and drug-resistance. Furthermore, AM404 suppressed the expression of FBXL5 E3-ligase, where AM404 sensitivity was mimicked by FBXL5-knockout. This study uncovers a new molecular mechanism for AM404-altering FBXL5 oncogene which mediates chemo-resistance and CRC invasion, thereby proposes to repurpose antibacterial AM404 as an anticancer agent

Deep Proteomic Analysis on Biobanked Paraffine-Archived Melanoma with Prognostic/Predictive Biomarker Read-Out

SIMPLE SUMMARY: Malignant melanoma is one of the most aggressive cancer types among the solid tumors; therefore, more clinically applicable protein biomarkers predicting survival and therapy response have mandatory importance, impacting patient treatment. The aim of the study was to discover new proteins in biobanked FFPE samples that relate to progression-free survival and response to targeted- and immuno-therapies in patients with melanoma. Protein expressions were detected and quantified by high-resolution mass spectrometry and were integrated with the clinical data and in-depth histopathology characterization. Sample groups with distinct protein expression profiles were connected to longer and shorter survival as well as other clinicopathologic features. In addition, key regulating proteins were assigned, as predictive of progression-free survival in immuno- and/or targeted therapy. Some of the proteins exhibited functionally important correlations to progression and therapy response, which ultimately contributes to a better understanding of melanoma pathology. ABSTRACT: The discovery of novel protein biomarkers in melanoma is crucial. Our introduction of formalin-fixed paraffin-embedded (FFPE) tumor protocol provides new opportunities to understand the progression of melanoma and open the possibility to screen thousands of FFPE samples deposited in tumor biobanks and available at hospital pathology departments. In our retrospective biobank pilot study, 90 FFPE samples from 77 patients were processed. Protein quantitation was performed by high-resolution mass spectrometry and validated by histopathologic analysis. The global protein expression formed six sample clusters. Proteins such as TRAF6 and ARMC10 were upregulated in clusters with enrichment for shorter survival, and proteins such as AIFI1 were upregulated in clusters with enrichment for longer survival. The cohort’s heterogeneity was addressed by comparing primary and metastasis samples, as well comparing clinical stages. Within immunotherapy and targeted therapy subgroups, the upregulation of the VEGFA-VEGFR2 pathway, RNA splicing, increased activity of immune cells, extracellular matrix, and metabolic pathways were positively associated with patient outcome. To summarize, we were able to (i) link global protein expression profiles to survival, and they proved to be an independent prognostic indicator, as well as (ii) identify proteins that are potential predictors of a patient’s response to immunotherapy and targeted therapy, suggesting new opportunities for precision medicine developments

Deep Functional Mapping For Predicting Cancer Outcome

The effective understanding of the biological behavior and prognosis of cancer subtypes is becoming very important in-patient administration. Cancer is a diverse disorder in which a significant medical progression and diagnosis for each subtype can be observed and characterized. Computer-aided diagnosis for early detection and diagnosis of many kinds of diseases has evolved in the last decade. In this research, we address challenges associated with multi-organ disease diagnosis and recommend numerous models for enhanced analysis. We concentrate on evaluating the Magnetic Resonance Imaging (MRI), Computed Tomography (CT), and Positron Emission Tomography (PET) for brain, lung, and breast scans to detect, segment, and classify types of cancer from biomedical images. Moreover, histopathological, and genomic classification of cancer prognosis has been considered for multi-organ disease diagnosis and biomarker recommendation. We considered multi-modal, multi-class classification during this study. We are proposing implementing deep learning techniques based on Convolutional Neural Network and Generative Adversarial Network. In our proposed research we plan to demonstrate ways to increase the performance of the disease diagnosis by focusing on a combined diagnosis of histology, image processing, and genomics. It has been observed that the combination of medical imaging and gene expression can effectively handle the cancer detection situation with a higher diagnostic rate rather than considering the individual disease diagnosis. This research puts forward a blockchain-based system that facilitates interpretations and enhancements pertaining to automated biomedical systems. In this scheme, a secured sharing of the biomedical images and gene expression has been established. To maintain the secured sharing of the biomedical contents in a distributed system or among the hospitals, a blockchain-based algorithm is considered that generates a secure sequence to identity a hash key. This adaptive feature enables the algorithm to use multiple data types and combines various biomedical images and text records. All data related to patients, including identity, pathological records are encrypted using private key cryptography based on blockchain architecture to maintain data privacy and secure sharing of the biomedical contents