Human Prostate Cancer Hallmarks Map

Human prostate cancer is a complex heterogeneous disease that mainly affects elder male population of the western world with a high rate of mortality. Acquisitions of diverse sets of hallmark capabilities along with an aberrant functioning of androgen receptor signaling are the central driving forces behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. These hallmark capabilities arise due to an intense orchestration of several crucial factors, including deregulation of vital cell physiological processes, inactivation of tumor suppressive activity and disruption of prostate gland specific cellular homeostasis. The molecular complexity and redundancy of oncoproteins signaling in prostate cancer demands for concurrent inhibition of multiple hallmark associated pathways. By an extensive manual curation of the published biomedical literature, we have developed Human Prostate Cancer Hallmarks Map (HPCHM), an onco-functional atlas of human prostate cancer associated signaling and events. It explores molecular architecture of prostate cancer signaling at various levels, namely key protein components, molecular connectivity map, oncogenic signaling pathway map, pathway based functional connectivity map etc. Here, we briefly represent the systems level understanding of the molecular mechanisms associated with prostate tumorigenesis by considering each and individual molecular and cell biological events of this disease process.


Figure 1. Human Prostate Cancer Hallmarks Map-A Hypothesis Driven Project.
(a) Prostate cancer at a glance-represents some basic facts around current prostate cancer treatment approach, its limitations and its associated post therapeutic complications. (b) Achilles' heel in cancer-depicts three important factors and their underlying features that most fundamentally challenges and dampens current molecularly targeted therapies in cancer. (c) 'Clausewitzian Dictum' in cancer biology-Here we propose for a multi target strategy, 'Clausewitzian Dictum' that essentially calls for a three level targeting-attack at the network level, attack at the cancer hallmark level, and for functional modular level. On the other hand, these three factors-cancer protein interactome network, cancer hallmark functional ability and its modular signaling network architecture together comprises the Achilles' heel in cancer. (d) Our Bottom Up Approach-Adaptor protein comprises our initial seed protein.
Binary protein interaction database [Human Protein Reference Database (HRPD)] provides information for adaptor protein and its physical interacting partner. For accessing the role of adaptor protein and its binary interacting partner in the context of prostate cancer, we manually curated the literature for each of adaptor protein and its corresponding interacting partner. Based on these information, we reconstruct adaptor centric here should be understood in terms of oncogenic robustness which arises as a typical consequence of functional redundancies and due to presence of alternative compensatory signaling circuitries as cancer has been previously depicted as a disease of pathways 3,9 . The emergence of treatment resistance in terms of chemo/radiation resistance 14,15 and castration resistance (CRPC) 7,8 additionally imparts the flexibilities of enemy's resisting ability.
In support of our hypothetical proposal, here we attempt to represent a comprehensive molecular framework for unraveling the complexities of human prostate cancer associated molecular mechanisms by structuring the existing knowledge. For concept of our work, please visit at-http://www.bioinformatics.org/canjovdb/concept.php

Human Prostate Cancer Hallmarks Map Aims & Features. Human Prostate Cancer Hallmarks Map
(HPCHM) is a manually constructed onco-functional atlas of human prostate tumorigenesis and is inherently based on classic cancer hallmarks concepts. It covers molecular signaling (both connectivity & pathway based) maps of most of the major cellular and pathogenic mechanisms implicated in prostate cancer progression, including 13 cancer hallmarks (10 classical & 3 prostate cancer unique hallmarks) and 11 cell biological feature based phenomenon (Table 1). It provides a comprehensive resource for revealing molecular architecture of human prostate cancer associated signaling at five specific level-first key molecular components of prostate cancer hallmarks & their underlying features ( Table 2), second prostate cancer hallmark and cell biological function based molecular connectivity map (Table 1), third hallmark based and its underlying events related oncogenic signaling pathway map (Table 3), fourth hallmark and underlying cell biological feature related pathway based functional connectivity map (Tables 4 and 5), fifth inter hallmark & cell biological feature based functional connectivity map (Tables 6 and 7). Human Prostate Cancer Hallmark Map (Fig. 1e) aims to represent the systems level understanding of prostate tumorigenesis, which can be accessed at-http://www.bioinformatics. org/canjovdb. By taking cellular signaling scaffolds or adaptor protein 16 as seed protein, we have reconstructed the corresponding adapter centric protein-protein binary interactome map in human prostate cancer with literature mined published experimental reports (Fig. 1d). This adapter protein centric molecular interactome provides the crucial 'scaffold' for gaining the holistic overview of prostate tumorigenesis in the context of its various hallmarks functioning. Apart from this, we have also incorporated information by key word searching (detailed in the material and methods section). For doing so, we have manually screened over 40 Hallmark-Insensitivity to antigrowth signals: prostate cancer cells become unresponsive to growth restraining signals. One of the fundamental features underlying the prostate tumorigenesis involves acquisition of insensitivity to growth inhibitory signals or evasion of growth suppression. The alterations in the functional behavior of diverse sets of tumor suppressor genes by various unfavorable circumstances such as deletion, mutation, loss of expression, rearrangement, promoter methylation and haplo-insufficiency greatly contributes in this acquisition process 1,17 . Apart from the actions of tumor suppressor, many aberrantly working cell proliferation negative regulators, deregulated cell cycle proteins, neutralization of pro-apoptotic stimulations and extracellular ATP also plays the key leading role in the establishment of insensitivity to antigrowth signaling during the course of tumorigenesis 1,18 . In HPCHM, systems medicine based view of insensitivity to antigrowth signaling hallmark consists of a core network of 88 proteins with 110 physical interactions, 14 pathway based functional modules and a signaling pathway map of 90 proteins, supported by information from 24 articles. The insensitivity to antigrowth signaling hallmark pathway map depicts 3 major signaling circuitries associated with cell growth & proliferation regulation, cell cycle regulation and suppression of apoptosis 1,4 . For holistic map of human prostate cancer specific insensitivity to anti growth signaling, visit -http://www.bioinformatics.org/canjovdb/hallmark.php?hall= H1.
Cell Biological Feature-Tumour Suppression. Suppression of tumor suppressive activity most critically aids in these acquisition process as progression of prostate tumorigenesis is closely associated with chromosomal rearrangement and alterations of multiple number of chromosomal regions including 1p, 6q, 7p, 7q, 8p, 10q, 13q, 16q, 17p, 17q and 18q, harboring the sites for many important tumor suppressors 19,20 . As a consequence of this molecular interactome in human prostate cancer. For gaining a functional organizational view, we then extracts various oncogenic signaling pathways maps, subnetworks and modules associated with diverse cell biological events and hallmark related phenomenon with other literature mined information's (detailed in the material and methods section). (e) Human Prostate Cancer Hallmarks Map Features-It provides a comprehensive resource for revealing molecular architecture of human prostate cancer at four specific levels namely, prostate cancer hallmark & its underlying feature based phenomenon, prostate cancer hallmark based and cell biological function based molecular connectivity map, hallmark based and its underlying events related oncogenic signaling pathway map, hallmark and inter hallmark related pathway based functional connectivity map.
Scientific RepoRts | 6:30691 | DOI: 10.1038/srep30691 loss of expression or functional inactivation of tumor suppressors, prostate cancer cell activates an intricate signaling circuitry that operates across diverse oncofunctional features associated signaling pathways. In HPCHM, systems medicine based view of tumor suppression contains a signaling pathway map of 93 proteins, supported by information from 45 articles. For holistic map of human prostate cancer associated tumor suppression, please visit at-http://www.bioinformatics.org/canjovdb/feature.php?feature= F1 Cell Biological Feature-Cell Cycle. Cell cycle deregulation plays an essential leading role in the suppression of anti-proliferative signaling as majorities of anti-proliferative signals are channeled through the proteins associated with cell cycle regulatory activities and G1 to S phase transition. All of the major cell cycle regulators in prostate cancer, which also responds to anti proliferative signaling, including p21Cip1, p27Kip1, Rb, p53 are deregulated by multiple ways and are subjected to loss of function 21 . Mutational inactivation that occurs during prostate tumorigenesis have been reported for p14, p16, p27, p53 and Rb genes 21 . In HPCHM, systems medicine based view of cell cycle contains a signaling pathway map of 75 proteins, supported by information from 36 articles. For holistic map of human prostate cancer specific cell cycle, please visit at-http://www.bioinformatics.org/ canjovdb/feature.php?featurety= P Hallmark-Self-sufficiency in growth signaling: prostate cancer cells produce their own growth signals to trigger cell growth, proliferation and survival. Three principal factors can be accounted for achieving the state of self-sufficiency in growth signaling or sustaining proliferative signaling in prostate cancer. The factors are, first, aberrant functioning of androgen receptor [22][23][24] , second, modulation of growth factor receptor activity 25 and third, modulation of an alternative intracellular signaling circuitries associated with cell growth, survival and proliferation 6,8,26 . Most strikingly, these factors also significantly contribute in the acquisition of androgen independent phenotypes and consequent emergence of castration resistant prostate cancer 27 . Androgen receptor mediated signaling, androgen independence and castration resistance can be considered as Prostate Cancer Unique Hallmark-Androgen Receptor (AR) mediated signaling: a nuclear receptor signaling axis that plays the most pivotal role in each and every steps of prostate tumorigenesis. Numerous molecular mechanisms have been proposed for shifting of AR activation from the balanced mode to promiscuous mode in castration resistant prostate cancer (CRPC). These molecular mechanisms can be basically grouped into three major categories-First, hyper activation of androgen receptor(AR) 22,23 as a result of mutation, amplification, increase in local synthesis of androgen, proteolysis, ligand independent activation and PTEN loss mediated nuclear exclusion of FOXO1 etc. Second, promiscuous activation of AR by overexpression of AR coactivators and down regulation of its corepressors 23 . Third, multiple alternative activation pathways mediated by transactivation 24 of AR. It has been proposed that a multitude of cellular signaling pathways including PI3K/AKT, ERK, SRC etc acts in concert and converge on AR for enhancing AR mediated non-genomic response and thereby impacting self-sustenance in growth signaling 22  and is largely responsible for castration resistant disease progression 22 . The growth of androgen independent castration resistant prostate tumor is intensified by up regulation of both autocrine and paracrine growth factors as a consequence of an adaptive response to androgen ablation and it essentially also fuels self sufficiency in growth signaling 25 . Apart from androgen ablation therapy, various factors and a series of oncogenic signaling events contributed for the acquisition of prostate cancer androgen independence 22 . In HPCHM, systems medicine based view of androgen independence contains a signaling pathway map of 143 proteins, supported by information from 54 articles. The androgen independence map is composed of six major pathway modules devoted to the mechanisms of prostate cancer androgen independence. The modules are associated with hypersensitive pathway, outlaw pathway, bypass pathway, co-activation pathway, promiscuous pathway and lurker cell pathway 22 Evasion of growth  suppression  90  10  14  -66  14  19  6  -24   Sustaning proliferative  signaling  91  6  8  12  65  17  3  14  30  36   Tumour promoting  inflammation  29  3  5  3  18  5  2  3  1  9 Cell death resistance 89 13   Cell Biological Feature-Cell proliferation. Deregulated cellular proliferation is an often considered hallmark feature of prostate tumorigenesis. Although the normal prostate epithelium is generally characterized by its low rate of cell proliferation, the prostatic intraepithelial neoplasia (PIN) and early invasive prostate carcinoma exhibits seven to ten fold increase in cell proliferation rate 28

Hallmark-Cell death resistance: prostate cancer cells resist and modulate apoptotic program.
Inhibition or evasion of apoptosis in comparison with enhanced cellular proliferation behaves as a critical pathophysiological factor for initiation and progression of majority of prostate adenocarcinomas 28,29

Hallmark-Angiogenesis: prostate cancer cells trigger the formation of new blood vessels.
Angiogenesis is needed for each and every steps in prostate tumorigenesis, ranging from tumor outgrowth to its metastatic dissemination 30,31 . The microvessel density in invasive tumors of human prostatic adenoma follows a direct correlation with tumors state, grade and its clinical courses 31  NF-κ B pathway and by locally producing diverse pro-inflammatory cytokines that supports the recruitment and activation of additional immune cells, such as macrophage, to the prostate 35 . In HPCHM, system medicine based view of inflammation hallmark comprises a core network of 23 proteins with 63 physical interactions, 16 pathway based functional modules and a signaling pathway map of 29 proteins, supported by information from 9 articles. The inflammation signaling pathway map represents four important signaling mechanisms that critically drives prostate tumour inflammation. The signaling mechanisms include modulation of NF-kB activation, modulation of inflammatory response, inflammasome activation and immune cell activation. For details, please visit at-http://www.bioinformatics.org/canjovdb/hallmark.php?hall= H3 Hallmark-Metabolic Reprogramming: prostate cancer cells deregulate metabolic program and energetics to support tumor formation. Although normal human prostatic epithelium cell growth rate critically balances its death rate but several factors including multivariate metabolic regulation by androgen receptor 36 , inactivation of tumor suppressors 37 and metabolic reprogramming 38 crucially fuels for the disruption of normal metabolic regulation, leading to an enhancement of prostate tumorigenesis. In HPCHM, systems medicine based view of metabolic reprogramming contains a signaling pathway map of 78 proteins, supported by information from 47 articles. The metabolic reprogramming oncogenic signaling map contains 12 critical metabolic switches that most significantly promotes prostate cancer cell metabolism. Hallmark-Enabling Replicative Immortality: prostate cancer cells have unlimited replicative potential. Telomere dysfunction and reactivation of the telomerase reverse transcriptase activity, which is typically associated with telomere maintenance, plays the profound significant roles in imparting limitless replicative potential of the prostate tumor cells 39,40 . Telomerase activity has not been detected in both normal prostate and benign prostatic hyperplastic tissue, but over 90% of prostate cancer exhibits significantly higher level of telomerase activity 40 . Although replicative senescence is assumed a significant impediment to cellular immortalization and transformation 1 , but the deregulation of a broad spectrum of senescence regulators greatly contributes in surpassing classical senescence program that eventually enhances prostate tumorigenesis. In HPCHM, systems medicine based view of replicative immortalization contains a signaling pathway map of 38 proteins, supported by information from 27 articles. The replicative immortalization signaling map represents detail molecular mechanisms associated with expression, modulation & enhancement of telomerase (hTERT) activity, down-regulation of replicative senescence and the role of androgen deprivation therapy in modulation of prostate tumors senescence program. For holistic map of prostate cancer replicative immortalization, visit-http://www.bioinformatics. org/canjovdb/hallmark.php?hall= H10 Hallmark-Avoidance of Immune Destruction: prostate cancer cells evade the monitoring capability of immune system. Tumor immune evasion is currently recognized as an emerging hallmark of cancer 1 and the current research strongly suggests that evasion of immune destruction plays a crucial role in the development of prostate cancer along with its pathogenic progression 41 . A significant alteration in cell mediated immune response is particularly observed during the course of prostate tumorigenesis and it involves multiple potential immune suppressive mechanisms 41,42 . In HPCHM, the avoidance of immune destruction holistic map represents an integrated view of signaling events between prostate cancer cell and various immune cells, which can be accessed at-http://www.bioinformatics.org/canjovdb/hallmark.php?hall= H9 Hallmark-Tumor Microenvironment: the surroundings of prostate tumor which critically fuels other hallmark manifestations. The tumor microenvironment in prostate cancer, which involves complex reciprocal interactions between prostate cancer epithelial cell and surrounding 'reactive stroma' , plays the most influential role in survival, angiogenesis, inflammation and its metastatic dissemination 43 . Prostate cancer 'reactive stroma' is typically characterized by the presence of cancer associated fibroblastic (CAF) and myofibroblastic cell populations along with a consequential loss of smooth muscle cells and is centrally responsible for the genesis of intratumoural heterogeneity 43,44 . Additionally, adipocytes play a very essential role in the modulation of prostate tumour microenvironment by secreting multiple growth factors (IGF1, TGF-beta, TNF-alpha, IL-6) and inflammatory adipokines 45 . In HPCHM, the tumor microenvironmental holistic map depicts the detail paracrine signaling interactions between prostate cancer cell and its surrounding 'reactive stroma' components including CAF, myofibroblast and adipocytes 1,43 . For details of prostate cancer associated tumor microenvironment, visit at-http://www.bioinformatics.org/canjovdb/hallmark.php?hall= H11

Discussion
Currently there is no effective molecularly targeted treatment strategy available for metastatic castration resistant prostate cancer 7,8 . Consequently, there is an actual need for identification of potential molecular targets and global molecular signaling map in the context of human prostate tumorigenesis, progression and treatment resistance 7,26 . But several challenging factors make the scenario frustratingly complex and practically infeasible. First, progression of prostate cancer involves a number of molecular and cellular processes at the same time 46 , second, prostate tumor's heterogeneous and multifocal nature 5,6 , third, prostate cancer's transition from androgen dependent to androgen unresponsive castration resistant form 7,8 . In addition, integrating information from multifarious sources of information across diverse systems is also a formidable challenge because of limitation of the available computational tools 2,47 . As a consequence, our understanding of the pathway signaling map and their modular interplay at prostate cancer hallmark level is largely lacking 4 . This is due to three principal reasons: first, although the classic cancer hallmark concept 1 alone provides the most fundamental organizing principles of neoplastic diseases, it remains descriptive 2 and no prostate cancer specific views are available yet 4,7,26 . Second, comprehensive information for most of cancer hallmark network in a generalized and global basis is largely unavailable there by restricting any computational based prediction 2 . Third, currently available pathway databases mainly offer the 'canonical pathway' based depiction, predominantly ignoring the facts that the multi stage process of tumorigenesis involves concomitantly multiple cellular processes and their signaling events 48 . For addressing all these issues, we have taken an initiative from a system medicine perspective for systems level exploration of prostate cancer associated molecular process and signaling cascades. Although the concept of system medicine offer an unique opportunity for simplification of complex systems (by transforming medicine into a network based platform in a context specific fashion) 47,49 , but it is assumed that the currently available interactome maps (generated by high-throughput methods) are more than 80% incomplete 50,47 . From this current standpoint, compilation of already available published experimental report in the literature provides a paramount opportunity for reconstruction of interactomes thereby inferring as realistic as possible views of molecular signaling maps underlying diseases 51 . To this line, literature mined binary protein interactome maps plays a potential role in attaining hypothesis about focused biological mechanisms 13,51 . In the current study, we have followed a bottom up approach, which is essentially based on network medicine 47,51 (Fig. 1d). Our effort reveals HPCHM, a web based contrivance, which provides the basic blue print of human prostate cancer specific 'hallmarkosme' , an assembly of cancer hallmarks and their underlying cell biological events. This 'hallmarkosome' represents a set of comprehensive molecular signaling map of each and individual cellular and molecular processes related with human prostate cancer pathogenesis. For comprehensive map of human prostate cancer associated hallmarks, please visit at the following link-http://www.bioinformatics.org/ canjovdb/comprehensive.php To our knowledge, Human Prostate Cancer Hallmarks Map (HPCHM) provides the first time depiction of global molecular signaling events that are underlying human prostate tumorigenesis. Several key features make the holistic maps unique in its representation: (1) It shows most of the crucial molecular signaling cascades, their crosstalks and regulatory molecular processes and also, 'a disease of pathway' based view that are usually deregulated during prostate cancer development and progression; (2) It covers all major human prostate tumour specific features including androgen dependent & independent signaling views, androgen receptor (AR) mediated signaling and their regulation, the role of prostate tumour specific modulators etc; (3) It presents detailed functional view of various onco & tumour suppressor proteins, whose collaborative action functions as one of the key driver of human prostate tumorigenesis; (4) It offers an inter hallmark connectivity map, as it has been currently hypothesized that cancer hallmarks are closely intertwined 2 .
In summary, here we represent a new frame work for gaining a comprehensive understanding of cellular and molecular events underlying of human prostate cancer pathogenesis by accumulating the existing knowledge. Most importantly, Human Prostate Cancer Hallmarks Map (HPCHM) provides a major step towards systems level mechanistic representation of prostate tumorigenesis that will surely give a new space for the development of novel therapeutic approaches.

Methods
Initial seed gene listing. Adapter protein comprises our initial seed gene. Human Protein Reference Database (HRPD) 52 provides information for different aspects of human proteins ranging from protein-protein interactions to molecular class information, post-translational modification to disease associations. From HRPD molecule class we got the list of 339 human adapter genes under the name of ' Adaptor molecule' . Literature Database searching. We manually curated the text for each of 339 adapter protein for their corresponding role in human prostate cancer. For literature search we used the NCBI PubMed database, for instance, by typing BCL2 in prostate cancer yields 159 different article containing experimentally verified information for the role of BCL2 adapter protein in prostate cancer. We manually curated each & individual of these articles for accessing the role of adapter proteins in prostate cancer.

Gene name normalization.
Additionally, an extensive literature survey was followed using NCBI Pubmed for retrieving articles related to human prostate cancer associated cell biological events by key word searching, for example using search terms 'prostate cancer cell proliferation' etc (corresponding cell biological events). The full text articles were manually screened for cell signaling, molecular interactions, oncogenic activation & tumor suppression processes, nuclear translocation events, transcriptional gene regulation and the hyperlinked PubMed identifier for the corresponding publication. For enhancing the accuracy of this textual curation, each of the information has been simultaneously cross verified three times by different researchers.
Binary Interaction partner selection. Adaptor proteins physical interacting partners were selected from the Human Protein Reference Database (HRPD) 52 . Each of the binary interacting partners is subjected for pubmed search in previously mentioned ways for identifying their role in human prostate cancer. In this manner, we constructed adaptor centric interactome map in prostate cancer based on proteins binary interaction along with literature curetted experimentally verified information.
Scientific RepoRts | 6:30691 | DOI: 10.1038/srep30691 Oncogenic Signaling Map extraction. We manually constructed the comprehensive pathway maps [Molecular Connectivity Map & Oncogenic Signaling Pathway Map] for prostate cancer hallmarks, their underlying cell biological events and its associated pathophenomic features (Androgen Independence, Castration Resistance etc). These maps are intrinsically based on the molecular interactions documented in adapter centric molecular interactome. In addition, we have also incorporated information from current existing publication on molecular events associated with pathogenesis and progression of human prostate cancer.

Pathway based Oncogenic Functional Connectivity Map reconstruction.
For pathway based information, we incorporated data from various sources, ranging from literature mined information to pathway databases, including KEGG 54 , Pathway Commons 55 and WikiPathways 56 through both manual curation and by using WEB-based GEne SeT AnaLysis Toolkit 57 . The subnetworks were derived by using Pajek 58 and represented in Pajek 58 and Cytoscape 59 based visualization platform.