Signaling, cancer cell plasticity, and intratumor heterogeneity.

Cancer's complexity is in part due to the presence of intratumor heterogeneity and the dynamic nature of cancer cell plasticity, which create substantial obstacles in effective cancer management. Variability within a tumor arises from the existence of diverse populations of cancer cells, impacting the progression, spread, and resistance to treatments. At the core of this variability is the concept of cellular plasticity - the intrinsic ability of cancer cells to alter their molecular and cellular identity in reaction to environmental and genetic changes. This adaptability is a cornerstone of cancer's persistence and progression, making it a formidable target for treatments. Emerging studies have emphasized the critical role of such plasticity in fostering tumor diversity, which in turn influences the course of the disease and the effectiveness of therapeutic strategies. The transformative nature of cancer involves a network of signal transduction pathways, notably those that drive the epithelial-to-mesenchymal transition and metabolic remodeling, shaping the evolutionary path of cancer cells. Despite advancements, our understanding of the precise molecular machinations and signaling networks driving these changes is still evolving, underscoring the necessity for further research. This editorial presents a series entitled "Signaling Cancer Cell Plasticity and Intratumor Heterogeneity" in Cell Communication and Signaling, dedicated to unraveling these complex processes and proposing new avenues for therapeutic intervention.

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

Transcending frontiers in prostate cancer: the role of oncometabolites on epigenetic regulation, CSCs, and tumor microenvironment to identify new therapeutic strategies.

Prostate cancer, as one of the most prevalent malignancies in males, exhibits an approximate 5-year survival rate of 95% in advanced stages. A myriad of molecular events and mutations, including the accumulation of oncometabolites, underpin the genesis and progression of this cancer type. Despite growing research demonstrating the pivotal role of oncometabolites in supporting various cancers, including prostate cancer, the root causes of their accumulation, especially in the absence of enzymatic mutations, remain elusive. Consequently, identifying a tangible therapeutic target poses a formidable challenge. In this review, we aim to delve deeper into the implications of oncometabolite accumulation in prostate cancer. We center our focus on the consequential epigenetic alterations and impacts on cancer stem cells, with the ultimate goal of outlining novel therapeutic strategies.

New Insights into Metabolic Alterations and Mitochondria Re-Arrangements in Pancreatic Adenocarcinoma.

Among the most aggressive cancer types, pancreatic ductal adenocarcinoma (PDAC) represents one with the highest lethality due to its resistance to therapies and to the frequent metastatic spread [...].

Mitochondrial Features of Mouse Myoblasts Are Finely Tuned by Low Doses of Ozone: The Evidence In Vitro.

The mild oxidative stress induced by low doses of gaseous ozone (O3) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O3 target. In this in vitro study, we investigated the mitochondrial response to low O3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O3 doses. The O3 concentration of 10 µg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 µg O3-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function.

Mitochondrial Dynamics as Potential Modulators of Hormonal Therapy Effectiveness in Males.

Worldwide the incidence of andrological diseases is rising every year and, together with it, also the interest in them is increasing due to their strict association with disorders of the reproductive system, including impairment of male fertility, alterations of male hormones production, and/or sexual function. Prevention and early diagnosis of andrological dysfunctions have long been neglected, with the consequent increase in the incidence and prevalence of diseases otherwise easy to prevent and treat if diagnosed early. In this review, we report the latest evidence of the effect of andrological alterations on fertility potential in both young and adult patients, with a focus on the link between gonadotropins' mechanism of action and mitochondria. Indeed, mitochondria are highly dynamic cellular organelles that undergo rapid morphological adaptations, conditioning a multitude of aspects, including their size, shape, number, transport, cellular distribution, and, consequently, their function. Since the first step of steroidogenesis takes place in these organelles, we consider that mitochondria dynamics might have a possible role in a plethora of signaling cascades, including testosterone production. In addition, we also hypothesize a central role of mitochondria fission boost on the decreased response to the commonly administrated hormonal therapy used to treat urological disease in pediatric and adolescent patients as well as infertile adults.

The rs1001179 SNP and CpG methylation regulate catalase expression in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an extremely variable clinical course. We have recently shown that high catalase (CAT) expression identifies patients with an aggressive clinical course. Elucidating mechanisms regulating CAT expression in CLL is preeminent to understand disease mechanisms and develop strategies for improving its clinical management. In this study, we investigated the role of the CAT promoter rs1001179 single nucleotide polymorphism (SNP) and of the CpG Island II methylation encompassing this SNP in the regulation of CAT expression in CLL. Leukemic cells harboring the rs1001179 SNP T allele exhibited a significantly higher CAT expression compared with cells bearing the CC genotype. CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-ß transcription factors. Moreover, CLL cells exhibited lower methylation levels than normal B cells, in line with the higher CAT mRNA and protein expressed by CLL in comparison with normal B cells. Methylation levels at specific CpG sites negatively correlated with CAT levels in CLL cells. Inhibition of methyltransferase activity induced a significant increase in CAT levels, thus functionally validating the role of CpG methylation in regulating CAT expression in CLL. Finally, the CT/TT genotypes were associated with lower methylation and higher CAT levels, suggesting that the rs1001179 T allele and CpG methylation may interact in regulating CAT expression in CLL. This study identifies genetic and epigenetic mechanisms underlying differential expression of CAT, which could be of crucial relevance for the development of therapies targeting redox regulatory pathways in CLL.

CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1.

AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5'-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates. Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

Quality of Life and Anorectal Malformations: A Single-Center Experience.

Purpose: The treatment and long term clinical outcomes of anorectal malformations (ARM) in children have always been the focus of pediatric surgeons. This study aimed at reporting our experience as far as long-term follow-up of ARM in children is concern. Methods: We enrolled patients treated between 1999 and 2019, and established selection criteria to choose appropriate subjects. A validated questionnaire was used to determine long-term quality of life outcomes. Results: Out of a total of 48 patients treated within the study period, 28 were enrolled in this study. Among the latter, more than 35% had at least one long-time complication, and more than 90% had a good lifestyle. Urinary and fecal continence was achieved in more than 95% of the patients using medical devices. Conclusion: This study aimed to bring up new concepts; taking into consideration all aspects of life in patients with ARM, from school life to sexuality, while evaluating fecal and urinary continence. This is essential for the improvement of the skills of the different specialists involved in the management of these patients, and for the implementation of strategies that can improve postoperative function. Most especially, it will also help improve communication between doctors to ensure an adequate transition of these children into adult life.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Divergent Roles of Mitochondria Dynamics in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors; it is often diagnosed at an advanced stage and is hardly treatable. These issues are strictly linked to the absence of early diagnostic markers and the low efficacy of treatment approaches. Recently, the study of the metabolic alterations in cancer cells has opened the way to important findings that can be exploited to generate new potential therapies. Within this scenario, mitochondria represent important organelles within which many essential functions are necessary for cell survival, including some key reactions involved in energy metabolism. These organelles remodel their shape by dividing or fusing themselves in response to cellular needs or stimuli. Interestingly, many authors have shown that mitochondrial dynamic equilibrium is altered in many different tumor types. However, up to now, it is not clear whether PDAC cells preferentially take advantage of fusion or fission processes since some studies reported a wide range of different results. This review described the role of both mitochondria arrangement processes, i.e., fusion and fission events, in PDAC, showing that a preference for mitochondria fragmentation could sustain tumor needs. In addition, we also highlight the importance of considering the metabolic arrangement and mitochondria assessment of cancer stem cells, which represent the most aggressive tumor cell type that has been shown to have distinctive metabolic features to that of differentiated tumor cells.

Testicular Torsion: Preliminary Results of In Vitro Cell Stimulation Using Chorionic Gonadotropin.

Testicular torsion is a pathology that occurs in young males generally before the age of 25. Despite surgery representing the only effective approach, there is still a need to identify a marker that can predict whether a preserved testicle will be functional. In addition, no therapeutic approach is currently considered in the post-operative phase. Through an approach based on the in vitro culture of a tissue strictly linked to the testicle, the gubernaculum, we defined the healthy state of the organ and the possible responsiveness to a therapy used in the andrology field, chorionic gonadotropin (hCG). Firstly, we optimized a protocol to obtain viable cells starting from a small piece of gubernacular tissue harvested during surgery with the aim to amplify cells in vitro. Intriguingly, only for a patient whose testicle had been removed during surgery due to an excessive necrotic area, gubernacular cells were not able to grow in culture. These data support the possibility of exploiting the gubernaculum to evaluate the healthy state of the testicle. Then, as we demonstrate that gubernacular cells express a luteinizing hormone receptor, to which hCG is specific, we analyzed the cellular response to hCG treatment on in vitro cultured cells derived from patients affected by testicular torsion. Our study opens the way for the possibility of evaluating testicle wellbeing after derotation through in vitro culture of a small piece of gubernaculum together with predicting the response to the treatment with hCG, which can have a positive effect on cell proliferation and vascularization.

A new frontier in therapy personalisation based on in vitro studies to preserve fertility potential of men.

At present, there is still a lack of attention to male infertility and fertility impairment. Indeed, the pathologies affecting the reproductive area in man are derived from anatomical or functional alterations of neuroendocrine system; thus, the study of these dysfunctions is necessary for a correct aetiopathogenetic and therapeutic framing of infertile patients. In this article, we underline the importance of the study of the molecular mechanisms regulated by the most common therapy used to treat infertile men, with the aim to highlight the necessity to avoid the administration of the wrong posology or, even more important, the wrong therapy to the patient. Accordingly, we present some pioneer data obtained on primary testicular cells cultured in vitro and treated with human chorionic gonadotropin (hCG). These data pave the way on the possibility to preliminarily test the effectiveness of the therapy in vitro, in order to identify the responsiveness of patient-derived cells to the treatment and its effectiveness in each subject, in order to identify the correct dosage in a personalised way.

Adolescent male genitalia dissatisfaction: a surgical perspective.

Genital dissatisfaction is well known in female and adults. Less is known about male adolescents and their genital satisfaction. The aim of this study was to investigate and report the role of surgery in male adolescents to improve the evaluation of their genitalia. We considered all patients treated for external genital pathology in the period of adolescence. Inclusion and exclusion criteria were created. Patients underwent an evaluation test before and after surgery. During the study period, 137 patients were treated, and at the end of the study, 98 cases were considered for analysis. The most frequent pathologies were webbed penis and penile curvature. A postoperative score improvement was noted and patients with concealed penis and webbed penis showed a better postoperative outcome. Overweight was considered an important factor associated with a worse preoperative score. Evaluation of the external genitalia is important in adolescents, and it is an understudied problem. Overweight may be associated with a worse evaluation of one's genital and should be clinically considered to avoid related social problems in adulthood. Therefore, cosmetic genital surgery should be considered even in male adolescents.

Integrated lipidomics and proteomics reveal cardiolipin alterations, upregulation of HADHA and long chain fatty acids in pancreatic cancer stem cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC.

Human Chorionic Gonadotropin-Mediated Induction of Breast Cancer Cell Proliferation and Differentiation.

Human chorionic gonadotropin (hCG) is a hormone that specifically binds to luteinizing hormone receptor (LHR) and exerts several roles, including the support of pregnancy and fetal gonadal steroidogenesis. Since hCG is also expressed by some tumor types, like breast cancer, many efforts have been made to study its role in neoplesia, with some studies showing a cancer-supportive role and others showing a cancer-protective role. A critical examination of the literature highlighted that the in vitro effect of hCG has been tested in the presence of fetal serum, which contains other gonadotropins, in the culture medium. Thus, we hypothesized that the use of serum in the cell culture medium might influence the cell response to the hCG treatment due to the presence of other hormones. Thus, we analyzed the in vitro effect of highly purified hCG on cell proliferation and the activation of the down-stream signal transduction pathway in three breast cancer cell lines, particularly focusing on MCF7, cultured in serum-deprived conditions. Our data show that hCG increases cell proliferation and activates the down-stream target Akt, together with a decrease of the LHR mRNA expression level. Finally, we also tested the differentiation capacity of hCG on MCF7 cancer stem cells (CSCs) and show that it favors the proliferation and differentiation of these cells, thus suggesting that hCG also renders cells more able to colonize and invade the organs.

Effects of CD20 antibodies and kinase inhibitors on B-cell receptor signalling and survival of chronic lymphocytic leukaemia cells.

Recently, clinical trial results have established inhibitors of B-cell receptor (BCR)-associated kinase (BAKi), with or without CD20 moniclonal antibodies (mAbs), as the preferred first-line treatment for most chronic lymphocytic leukaemia (CLL) patients. Using phosphospecific flow cytometry, we showed that in leukaemic cells from CLL patients the CD20 therapeutic antibodies - rituximab, ofatumumab, and obinutuzumab - inhibited BCR signalling pathways targeting preferentially pBTKY551 - but not BTKY223 - and pAKT. On the contrary, ibrutinib and idelalisib reduced pBTKY223 to a higher extent than pBTKY551 . The strong reduction of pAKT induced by idelalisib was enhanced by its combination with rituximab or ofatumumab. Moreover, CD20 mAbs and BAKi induced the death of leukaemia cells that was significantly potentiated by their combination. Analysis of the enhancement of cell death in these combinations revealed an approximately additive enhancement induced by rituximab or obinutuzumab combined with ibrutinib or idelalisib. Taken together, our data identified negative regulatory effects of CD20 mAbs and their combinations with BAKi on BCR signalling and cell survival in CLL. In conclusion, this study advances our understanding of mechanisms of action of CD20 mAbs as single agents or in combination with BAKi and could inform on the potential of combined therapies in ongoing and future clinical trials in patients with CLL.

Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. METHODS: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. RESULTS: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters' expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. CONCLUSION: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.