Differential Proteomic Analysis of Human Sperm: A Systematic Review to Identify Candidate Targets to Monitor Sperm Quality.

PURPOSE: The advent of proteomics provides new opportunities to investigate the molecular mechanisms underlying male infertility. The selection of relevant targets based on a single analysis is not always feasible, due to the growing number of proteomic studies with conflicting results. Thus, this study aimed to systematically review investigations comparing the sperm proteome of normozoospermic and infertile men to define a panel of proteins with the potential to be used to evaluate sperm quality. MATERIALS AND METHODS: A literature search was conducted on PubMed, Web of Science, and Scopus databases following the PRISMA guidelines. To identify proteins systematically reported, first the studies were divided by condition into four groups (asthenozoospermia, low motility, unexplained infertility, and infertility related to risk factors) and then, all studies were analysed simultaneously (poor sperm quality). To gain molecular insights regarding identified proteins, additional searches were performed within the Human Protein Atlas, Mouse Genome Informatics, UniProt, and PubMed databases. RESULTS: Thirty-two studies were included and divided into 4 sub-analysis groups. A total of 2752 proteins were collected, of which 38, 1, 3 and 2 were indicated as potential markers for asthenozoospermia, low motility, unexplained infertility and infertility related to risk factors, respectively, and 58 for poor sperm quality. Among the identified proteins, ACR, ACRBP, ACRV1, ACTL9, AKAP4, ATG3, CCT2, CFAP276, CFAP52, FAM209A, GGH, HPRT1, LYZL4, PRDX6, PRSS37, REEP6, ROPN1B, SPACA3, SOD1, SPEM1, SPESP1, SPINK2, TEKT5, and ZPBP were highlighted due to their roles in male reproductive tissues, association with infertility phenotypes or participation in specific biological functions in spermatozoa. CONCLUSIONS: Sperm proteomics allows the identification of protein markers with the potential to overcome limitations in male infertility diagnosis and to understand changes in sperm function at the molecular level. This study provides a reliable list of systematically reported proteins that could be potential targets for further basic and clinical studies.

Protein Mimicry and the Design of Bioactive Cell-Penetrating Peptides: The Genesis of STOPSPERM Bioportides.

The mature spermatozoon, a highly differentiated cell equipped for the sole purpose of fertilization, lacks the protein machinery required for conventional endocytotic mechanisms. Perhaps contrary to expectation, cell-penetrating peptides (CPPs) rapidly translocate across the unique sperm plasma membrane to accrete within distinct intracellular compartments. Confocal microscopy, employing red-fluorescent CPPs and bioportides, is a convenient platform to study this membrane translocation process. In the virtual absence of genetic expression, rapid physiological responses of human sperm are dependent upon protein-protein interactions that may be regulated by posttranslational modifications including phosphorylation. This chapter provides an outline of the design of bioactive CPPs, or bioportides, which include protein-mimetic sequences from the interaction domains of sperm proteins. Protocols are included which enable the biological assessment of the impact of bioportides upon the viability and motility of spermatozoa.

Bioinformatic Approach to Unveil Key Differentially Expressed Proteins in Human Sperm After Slow and Rapid Cryopreservation.

Currently, two conventional freezing techniques are used in sperm cryopreservation: slow freezing (SF) and rapid freezing (RF). Despite the protocolar improvements, cryopreservation still induces significant alterations in spermatozoon that are poorly understood. Here, available proteomic data from human cryopreserved sperm was analyzed through bioinformatic tools to unveil key differentially expressed proteins (DEPs) that can be used as modulation targets or quality markers. From the included proteomic studies, 160 and 555 DEPs were collected for SF and RF groups, respectively. For each group, an integrative network was constructed using gene ontology and protein-protein interaction data to identify key DEPs. Among them, arylsulfatase A (ARSA) was highlighted in both freezing networks, and low ARSA levels have been associated with poor-sperm quality. Thus, ARSA was selected for further experimental investigation and its levels were assessed in cryopreserved samples by western blot. ARSA levels were significantly decreased in RF and SF samples (∼31.97 and ∼39.28%, respectively). The bioinformatic analysis also revealed that the DEPs were strongly associated with proteasomal and translation pathways. The purposed bioinformatic approach allowed the identification of potential key DEPs in freeze-thawed human spermatozoa. ARSA has the potential to be used as a marker to assess sperm quality after cryopreservation.

Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility.

OBJECTIVE: To design protein phosphatase 1 (PP1)-disrupting peptides covalently coupled to inert cell-penetrating peptides (CPPs) as sychnologically organized bioportide constructs as a strategy to modulate sperm motility. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S)/ANIMAL(S): Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Effect of the bioportides on the activity and interactions of PP1γ2-a PP1 isoform expressed exclusively in testicular germ cells and sperm-and on sperm vitality and motility. RESULT(S): PP1-disrupting peptides were designed based on the sequences from: 1) a sperm-specific PP1 interactor (A kinase anchor protein 4); and 2) a PP1 inhibitor (protein phosphatase inhibitor 2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact on PP1γ2 activity and sperm motility. Molecular modeling studies further facilitated the identification of an optimized PP1-binding sequence and enabled the development of a modified stop-sperm bioportide with reduced size and increased potency of action. In addition, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome. CONCLUSION(S): These investigations demonstrate the utility of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon spermatozoa motility, a key prognostic indicator of male fertility.

Investigation of spectroscopic and proteomic alterations underlying prostate carcinogenesis.

Prostate cancer (PCa) treatment remains challenging, especially in advanced stages, where the lack of sensitivity and specificity of available biomarkers makes it difficult to establish an accurate prognosis. Therefore, it is imperative to study PCa biology to identify key molecules that can improve PCa management. In this study, eight prostate tumor tissues and paired normal tissues were analyzed using two approaches-Fourier-transform infrared (FT-IR) spectroscopy for spectroscopic profiling of biomolecules and antibody microarray for signaling proteins-with the main goal of identifying metabolic and proteomic changes that enable the distinction between normal and tumor conditions. Principal component analysis of FT-IR spectra revealed different spectroscopic signals for each condition. The most relevant changes in prostate tumor tissues identified by FT-IR were dysregulation in lipid metabolism, lower polysaccharide and glycogen content, higher nucleic acid content, and increased protein phosphorylation. Using an antibody microarray, 42 proteins were identified as differentially regulated between the two conditions; 14 of those revealed changes in their phosphorylation status. These proteins include transcription factors and kinases and constitute a highly-interconnected interaction network. Altogether, our data reveal metabolic and proteomic alterations that may be of interest in future translational studies aimed at establishing PCa prognosis and treatment. SIGNIFICANCE: Prostate tumor tissues and adjacent benign tissues were analyzed using two approaches-Fourier-transform infrared (FT-IR) spectroscopy for biomolecules and an antibody microarray for signaling proteins, which allowed to identify a panel of metabolic and proteomic alterations that may be of interest in future translational studies to enable the distinction between normal and tumor conditions.

First Insights on the Presence of the Unfolded Protein Response in Human Spermatozoa.

The unfolded protein response (UPR) is involved in protein quality control and is activated in response to several stressors. Although in testis the UPR mechanisms are well described, their presence in spermatozoa is contentious. We aimed to investigate the presence of UPR-related proteins in human sperm and the impact of oxidative stress induction in UPR activation. To identify UPR-related proteins in human sperm, a bioinformatic approach was adopted. To explore the activation of UPR, sperm were exposed to hydrogen peroxide (H2O2) and motility, vitality, and the levels of UPR-related proteins were assessed. We identified 97 UPR-related proteins in human sperm and showed, for the first time, the presence of HSF1, GADD34, and phosphorylated eIF2α. Additionally, the exposure of human sperm to H2O2 resulted in a significant decrease in sperm viability and motility and an increase in the levels of HSF1, HSP90, HSP60, HSP27, and eIF2α; all proteins involved in sensing and response to unfolded proteins. This study gave us a first insight into the presence of UPR mechanisms in the male gamete. However, the belief that sperm are devoid of transcription and translation highlight the need to clarify if these pathways are activated in sperm in the same way as in somatic cells.

mTOR Signaling Pathway Regulates Sperm Quality in Older Men.

Understanding how age affects fertility becomes increasingly relevant as couples delay childbearing toward later stages of their lives. While the influence of maternal age on fertility is well established, the impact of paternal age is poorly characterized. Thus, this study aimed to understand the molecular mechanisms responsible for age-dependent decline in spermatozoa quality. To attain it, we evaluated the impact of male age on the activity of signaling proteins in two distinct spermatozoa populations: total spermatozoa fraction and highly motile/viable fraction. In older men, we observed an inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) in the highly viable spermatozoa population. On the contrary, when considering the entire spermatozoa population (including defective/immotile/apoptotic cells) our findings support an active mTORC1 signaling pathway in older men. Additionally, total spermatozoa fractions of older men presented increased levels of apoptotic/stress markers (e.g., cellular tumor antigen p53-TP53) and mitogen-activated protein kinases (MAPKs) activity. Moreover, we established that the levels of most signaling proteins analyzed were consistently and significantly altered in men more than 27 years of age. This study was the first to associate the mTOR signaling pathway with the age impact on spermatozoa quality. Additionally, we constructed a network of the sperm proteins associated with male aging, identifying TP53 as a central player in spermatozoa aging.

Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

"A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. SIGNIFICANCE: Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of bioinformatics tools to their full potential to analyse protein-protein interactions as a comprehensive network.

Study on the short-term effects of increased alcohol and cigarette consumption in healthy young men's seminal quality.

Many studies have reported a negative impact of lifestyle factors on testicular function, spermatozoa parameters and pituitary-gonadal axis. However, conclusions are difficult to draw, since studies in the general population are rare. In this study we intended to address the early and late short-term impact of acute lifestyle alterations on young men's reproductive function. Thirty-six healthy male students, who attended the Portuguese academic festivities, provided semen samples and answered questionnaires at three time-points. The consumption of alcohol and cigarette increased more than 8 and 2 times, respectively, during the academic festivities and resulted in deleterious effects on semen quality: one week after the festivities, a decrease on semen volume, spermatozoa motility and normal morphology was observed, in parallel with an increase on immotile spermatozoa, head and midpiece defects and spermatozoa oxidative stress. Additionally, three months after the academic festivities, besides the detrimental effect on volume, motility and morphology, a negative impact on spermatozoa concentration was observed, along with a decrease on epididymal, seminal vesicles and prostate function. This study contributed to understanding the pathophysiology underlying semen quality degradation induced by acute lifestyle alterations, suggesting that high alcohol and cigarette consumption are associated with decreased semen quality in healthy young men.

Construction and analysis of a human testis/sperm-enriched interaction network: Unraveling the PPP1CC2 interactome.

BACKGROUND: Phosphoprotein phosphatase 1 catalytic subunit gamma 2 (PPP1CC2), a PPP1CC tissue-specific alternative splice restricted to testicular germ cells and spermatozoa, is essential for spermatogenesis and spermatozoa motility. The key to understand PPP1CC2 regulation lies on the characterization of its interacting partners. METHODS: We construct a testis/sperm-enriched protein interaction network and analyzed the topological properties and biological context of the network. Further the interaction of a potential target for pharmacological intervention was validated in human spermatozoa. RESULTS: A total of 1778 proteins and 32,187 interactions between them were identified in the testis/sperm-enriched network. The network analysis revealed the members of functional modules that interact more tightly with each other. In the network, PPP1CC was located in the fourth maximum core part (k=41) and had 106 direct interactors. Sixteen PPP1CC interactors were involved in spermatogenesis-related categories. Also, PPP1CC had 50 direct interactors, highly interconnected and many of them part of the network maximum core (k=44), associated with motility-related annotations, including several previously uncharacterized interactors, such as, LMNA, JAK2 and RIPK3. CONCLUSIONS: In this study we integrated tissue-specific protein expression and protein-protein interaction data in order to identify key PPP1CC2 complexes for male reproductive functions. One of the most intriguing interactors was A-kinase anchor protein 4 (AKAP4), a testis-specific protein related to infertility phenotypes and involved in all major motility-related annotations. GENERAL SIGNIFICANCE: We demonstrated for the first time the interaction between PPP1CC2 and AKAP4 in human spermatozoa and the potential of the complex as contraceptive target.

Non-stop lab week: A real laboratory experience for life sciences postgraduate courses.

At the Portuguese universities, practical classes of life sciences are usually professor-centered 2-hour classes. This approach results in students underprepared for a real work environment in a research/clinical laboratory. To provide students with a real-life laboratory environment, the Non-Stop Lab Week (NSLW) was created in the Molecular Biomedicine master program at the University of Aveiro, Portugal. The unique feature of the NSLW is its intensity: during a 1-week period, students perform a subcloning and a protein expression project in an environment that mimics a real laboratory. Students work autonomously, and the progression of work depends on achieving the daily goals. Throughout the three curricular years, most students considered the intensity of the NSLW a very good experience and fundamental for their future. Moreover, after some experience in a real laboratory, students state that both the techniques and the environment created in the NSLW were similar to what they experience in their current work situation. The NSLW fulfills a gap in postgraduate students' learning, particularly in practical skills and scientific thinking. Furthermore, the NSLW experience provides skills to the students that are crucial to their future research area. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:297-303, 2016.

Characterisation of several ankyrin repeat protein variant 2, a phosphoprotein phosphatase 1-interacting protein, in testis and spermatozoa.

Phosphoprotein phosphatase 1 (PPP1) catalytic subunit gamma 2 (PPP1CC2), a PPP1 isoform, is largely restricted to testicular germ cells and spermatozoa. The key to understanding PPP1 regulation in male germ cells lies in the identification and characterisation of its interacting partners. This study was undertaken to determine the expression patterns of the several ankyrin repeat protein variant 2 (SARP2), a PPP1-interacting protein, in testis and spermatozoa. SARP2 was found to be highly expressed in testis and spermatozoa, and its interaction with human spermatozoa endogenous PPP1CC2 was confirmed by immunoprecipitation. Expression analysis by RT-qPCR revealed that SARP2 and PPP1CC2 mRNA levels were significantly higher in the spermatocyte fraction. However, microscopy revealed that SARP2 protein was only present in the nucleus of elongating and mature spermatids and in spermatozoa. In spermatozoa, SARP2 was prominently expressed in the connecting piece and flagellum, as well as, to a lesser extent, in the acrosome. A yeast two-hybrid approach was used to detect SARP2-interacting proteins and a relevant interaction with a novel sperm-associated antigen 9 (SPAG9) variant, a testis and spermatozoa-specific c-Jun N-terminal kinase-binding protein, was validated in human spermatozoa. Given the expression pattern of SARP2 and its association with PPP1CC2 and SPAG9, it may play a role in spermiogenesis and sperm function, namely in sperm motility and the acrosome reaction.

Oxidative stress markers: Can they be used to evaluate human sperm quality?

OBJECTIVE: To study the effects of an acute lifestyle change in human semen oxidative stress (OS) by applying seminal parameters and OS markers and to study the feasibility of mid-infrared spectroscopy with Fourier transform infrared spectroscopy (FT-IR) as a complementary tool to evaluate the effects of OS on human sperm samples. MATERIAL AND METHODS: Sperm samples were collected from healthy male students (n=8) who voluntarily submitted themselves to acute lifestyle changes during academic festivities. The samples were obtained before and after the academic festivities and were compared by basic semen analyses and OS markers, namely with thiobarbituric acid reactive species (TBARS) and total thiol (SH) groups by spectrophotometric assays and carbonyl (CO) groups by slot blot. The samples were also submitted for spectroscopic analysis to evaluate the feasibility of FT-IR coupled with multivariate analysis to calibrate OS biomarkers. Statistical analysis was performed applying paired Wilcoxon tests. RESULTS: Acute lifestyle alterations during academic week festivities were associated with a significant decrease in the percentage of normal spermatozoa in the ejaculate (p=0.011) and a decrease in sperm concentration and in semen volume. Regarding OS, acute lifestyle changes promoted a significant increment of TBARS (p=0.018) and an increasing trend in the SH group. With FT-IR and multivariate analysis, it was possible to develop calibration models to the following protein OS biomarkers: SH groups and CO. CONCLUSIONS: Acute lifestyle changes during academic festivities have negative effects on sperm quality, in both conventional seminal parameters and OS markers. The evaluation of OS biomarkers and FT-IR could improve andrology diagnosis and therapeutic follow-up.

Profiling signaling proteins in human spermatozoa: biomarker identification for sperm quality evaluation.

OBJECTIVE: To determine the correlation between semen basic parameters and the expression and activity of signaling proteins. DESIGN: In vitro studies with human spermatozoa. SETTING: Academic research institute. PATIENT(S): Thirty-seven men provided semen samples for routine analysis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Basic semen parameters tracked included sperm DNA fragmentation (SDF), the expression levels of 75 protein kinases, and the phosphorylation/cleavage patterns of 18 signaling proteins in human spermatozoa. RESULT(S): The results indicated that the phosphorylated levels of several proteins (Bad, GSK-3ß, HSP27, JNK/SAPK, mTOR, p38 MAPK, and p53), as well as cleavage of PARP (at D214) and Caspase-3 (at D175), were significantly correlated with motility parameters. Additionally, the percentage of morphologically normal spermatozoa demonstrated a significant positive correlation with the phosphorylated levels of p70 S6 kinase and, in turn, head defects and the teratozoospermia index (TZI) showed a significant negative correlation with the phosphorylated levels of Stat3. There was a significant positive correlation between SDF and the teratozoospermia index, as well as the presence of head defects. In contrast, SDF negatively correlated with the percentage of morphologically normal spermatozoa and the phosphorylation of Akt and p70 S6 kinase. Subjects with varicocele demonstrated a significant negative correlation between head morphological defects and the phosphorylated levels of Akt, GSK3ß, p38 MAPK, and Stat1. Additionally, 34 protein kinases were identified as expressed in their total protein levels in normozoospermic samples. CONCLUSION(S): This study contributed toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters.

Signalling pathways involved in oocyte growth, acquisition of competence and activation.

The oocyte's primary function is to be fertilised by a spermatozoon in order to create a viable embryo. Oocyte growth and development are initiated during embryogenesis and occur in parallel to follicular development. Factors produced by the oocyte bind to receptors on follicular cells, ensuring follicular development. Oocytes begin meiosis during foetal development and are arrested in prophase I by elevated levels of cyclic adenosine monophosphate (cAMP). Activation of mitogen-activated protein kinases triggers degradation of cAMP, allowing oocyte maturation to proceed. The production of progesterone and prostaglandins during the ovulation process ultimately activates proteases, whose action helps to release the oocyte into the Fallopian tube. Oocyte activation depends on fertilisation and is induced by changes in intracellular calcium levels. Dysregulation of these pathways is involved in the pathogenesis of several diseases including the syndrome of oocyte maturation failure.

The power of the yeast two-hybrid system in the identification of novel drug targets: building and modulating PPP1 interactomes.

Since the description of the yeast two-hybrid (Y2H) method, it has become more and more evident that it is the most commonly used method to identify protein-protein interactions (PPIs). The improvements in the original Y2H methodology in parallel with the idea that PPIs are promising drug targets, offer an excellent opportunity to apply the principles of this molecular biology technique to the pharmaceutical field. Additionally, the theoretical developments in the networks field make PPI networks very useful frameworks that facilitate many discoveries in biomedicine. This review highlights the relevance of Y2H in the determination of PPIs, specifically phosphoprotein phosphatase 1 interactions, and its possible outcomes in pharmaceutical research.

Amyloid precursor protein interaction network in human testis: sentinel proteins for male reproduction.

BACKGROUND: Amyloid precursor protein (APP) is widely recognized for playing a central role in Alzheimer's disease pathogenesis. Although APP is expressed in several tissues outside the human central nervous system, the functions of APP and its family members in other tissues are still poorly understood. APP is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. Furthermore, APP superfamily members are known to be associated with fertility. Knowledge on the protein networks of APP in human testis and spermatozoa will shed light on the function of APP in the male reproductive system. RESULTS: We performed a Yeast Two-Hybrid screen and a database search to study the interaction network of APP in human testis and sperm. To gain insights into the role of APP superfamily members in fertility, the study was extended to APP-like protein 2 (APLP2). We analyzed several topological properties of the APP interaction network and the biological and physiological properties of the proteins in the APP interaction network were also specified by gene ontologyand pathways analyses. We classified significant features related to the human male reproduction for the APP interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility. CONCLUSIONS: The present work provides the first report on the APP interactome in human testis. Our approach allowed the identification of novel interactions and recognition of key APP interacting proteins for male reproduction, particularly in sperm-oocyte interaction.

TGF-ß cascade regulation by PPP1 and its interactors -impact on prostate cancer development and therapy.

Protein phosphorylation is a key mechanism by which normal and cancer cells regulate their main transduction pathways. Protein kinases and phosphatases are precisely orchestrated to achieve the (de)phosphorylation of candidate proteins. Indeed, cellular health is dependent on the fine-tune of phosphorylation systems, which when deregulated lead to cancer. Transforming growth factor beta (TGF-ß) pathway involvement in the genesis of prostate cancer has long been established. Many of its members were shown to be hypo- or hyperphosphorylated during the process of malignancy. A major phosphatase that is responsible for the vast majority of the serine/threonine dephosphorylation is the phosphoprotein phosphatase 1 (PPP1). PPP1 has been associated with the dephosphorylation of several proteins involved in the TGF-ß cascade. This review will discuss the role of PPP1 in the regulation of several TGF-ß signalling members and how the subversion of this pathway is related to prostate cancer development. Furthermore, current challenges on the protein phosphatases field as new targets to cancer therapy will be addressed.

Prostate cancer: the need for biomarkers and new therapeutic targets.

Prostate cancer (PCa) incidence and mortality have decreased in recent years. Nonetheless, it remains one of the most prevalent cancers in men, being a disquieting cause of men's death worldwide. Changes in many cell signaling pathways have a predominant role in the onset, development, and progression of the disease. These include prominent pathways involved in the growth, apoptosis, and angiogenesis of the normal prostate gland, such as androgen and estrogen signaling, and other growth factor signaling pathways. Understanding the foundations of PCa is leading to the discovery of key molecules that could be used to improve patient management. The ideal scenario would be to have a panel of molecules, preferably detectable in body fluids, that are specific and sensitive biomarkers for PCa. In the early stages, androgen deprivation is the gold standard therapy. However, as the cancer progresses, it eventually becomes independent of androgens, and hormonal therapy fails. For this reason, androgen-independent PCa is still a major therapeutic challenge. By disrupting specific protein interactions or manipulating the expression of some key molecules, it might be possible to regulate tumor growth and metastasis formation, avoiding the systemic side effects of current therapies. Clinical trials are already underway to assess the efficacy of molecules specially designed to target key proteins or protein interactions. In this review, we address that recent progress made towards understanding PCa development and the molecular pathways underlying this pathology. We also discuss relevant molecular markers for the management of PCa and new therapeutic challenges.