New cellular imaging-based method to distinguish the SPG4 subtype of hereditary spastic paraplegia.

BACKGROUND AND PURPOSE: Microtubule defects are a common feature in several neurodegenerative disorders, including hereditary spastic paraplegia. The most frequent form of hereditary spastic paraplegia is caused by mutations in the SPG4/SPAST gene, encoding the microtubule severing enzyme spastin. To date, there is no effective therapy available but spastin-enhancing therapeutic approaches are emerging; thus prognostic and predictive biomarkers are urgently required. METHODS: An automated, simple, fast and non-invasive cell imaging-based method was developed to quantify microtubule cytoskeleton organization changes in lymphoblastoid cells and peripheral blood mononuclear cells. RESULTS: It was observed that lymphoblastoid cells and peripheral blood mononuclear cells from individuals affected by SPG4-hereditary spastic paraplegia show a polarized microtubule cytoskeleton organization. In a pilot study on freshly isolated peripheral blood mononuclear cells, our method discriminates SPG4-hereditary spastic paraplegia from healthy donors and other hereditary spastic paraplegia subtypes. In addition, it is shown that our method can detect the effects of spastin protein level changes. CONCLUSIONS: These findings open the possibility of a rapid, non-invasive, inexpensive test useful to recognize SPG4-hereditary spastic paraplegia subtype and evaluate the effects of spastin-enhancing drug in non-neuronal cells.

The Cockayne syndrome group A and B proteins are part of a ubiquitin-proteasome degradation complex regulating cell division.

Cytokinesis is monitored by a molecular machinery that promotes the degradation of the intercellular bridge, a transient protein structure connecting the two daughter cells. Here, we found that CSA and CSB, primarily defined as DNA repair factors, are located at the midbody, a transient structure in the middle of the intercellular bridge, where they recruit CUL4 and MDM2 ubiquitin ligases and the proteasome. As a part of this molecular machinery, CSA and CSB contribute to the ubiquitination and the degradation of proteins such as PRC1, the Protein Regulator of Cytokinesis, to ensure the correct separation of the two daughter cells. Defects in CSA or CSB result in perturbation of the abscission leading to the formation of long intercellular bridges and multinucleated cells, which might explain part of the Cockayne syndrome phenotypes. Our results enlighten the role played by CSA and CSB as part of a ubiquitin/proteasome degradation process involved in transcription, DNA repair, and cell division.

HOPS/TMUB1 retains p53 in the cytoplasm and sustains p53-dependent mitochondrial apoptosis.

Apoptotic signalling by p53 occurs at both transcriptional and non-transcriptional levels, as p53 may act as a direct apoptogenic stimulus via activation of the intrinsic mitochondrial pathway. HOPS is a highly conserved, ubiquitously expressed shuttling protein with an ubiquitin-like domain. We generated Hops-/- mice and observed that they are viable with no apparent phenotypic defects. However, when treated with chemotherapeutic agents, Hops-/- mice display a significant reduction in apoptosis, suggesting an impaired ability to respond to genotoxic stressors. We show that HOPS acts as a regulator of cytoplasmic p53 levels and function. By binding p53, HOPS inhibits p53 proteasomal degradation and favours p53 recruitment to mitochondria and apoptosis induction. By interfering with importin α, HOPS further increases p53 cytoplasmic levels. Thus, HOPS promotes the p53-dependent mitochondrial apoptosis pathway by preserving cytoplasmic p53 from both degradation and nuclear uptake.

HIPK2 controls cytokinesis and prevents tetraploidization by phosphorylating histone H2B at the midbody.

Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.

Mutant cohesin drives chromosomal instability in early colorectal adenomas.

Chromosome missegregation leads to chromosomal instability (CIN), thought to play a role in cancer development. As cohesin functions in guaranteeing correct chromosome segregation, increasing data suggest its involvement in tumorigenesis. In a screen of a large series of early colorectal adenomas, a precocious step during colorectal tumorigenesis, we identified 11 mutations in SMC1A core cohesin subunit. In addition, we sequenced the SMC1A gene in colorectal carcinomas and we found only one mutation. Finally, the transfection of the SMC1A mutations identified in early adenomas and wild-type SMC1A gene silencing in normal human fibroblasts led to CIN. Our findings that SMC1A mutations decrease from early adenomas to colorectal cancers and that mutations lead to CIN suggest that mutant cohesin could play a pivotal role during colorectal cancer development.

Prognostic role of serum p53 antibodies in lung cancer.

BACKGROUND: Mutations in the TP53 (Tumour Protein 53) gene can lead to expression of mutant p53 proteins that accumulate in cancer cells and can induce circulating p53 antibodies in cancer patients. Our aim was to evaluate the presence and prognostic role of these antibodies in lung cancer patients and to investigate whether they were related to p53 expression or TP53 mutations in tumour tissues. METHODS: A total of 201 lung cancer patients were evaluated for p53 antibodies by ELISA (Enzyme-Linked Immunosorbent Assay) and control was obtained from 54 patients with non-malignant disorders; p53 expression was evaluated in 131 of the lung cancer patients by immunohistochemistry and TP53 mutations were then investigated in 53 tumours positively staining for p53 and in 12 tumours without p53 overexpression, whose DNA was available for direct sequencing. RESULTS: Our results show that 20.4% of cancer patients have positive levels of p53 antibodies, while none of the controls resulted positive. High levels of p53 expression are detected in 57.3% of cases and a significant correlation between serum p53 antibodies and high levels of p53 expression in the corresponding tumours is observed. In non-small cell lung cancer, p53 antibodies are significantly associated with poorly differentiated tumours; furthermore, high levels of p53 expression significantly correlated with squamous cell carcinoma and tumours with highest grade. Survival time of non-small cell lung cancer patients low/negative for serum p53 antibodies was significantly longer compared to patients with positive levels (p = 0.049); in particular, patients with squamous cell carcinoma, but not adenocarcinoma, low/negative for these antibodies show a significant better survival compared to serum-positive patients (p = 0.044). CONCLUSIONS: In our study, detection of serum p53 antibodies in non-small cell lung cancer patients has been shown to be useful in identifying subsets of patients with poor prognosis. A significant correlation between the presence of serum p53 antibodies in lung cancer patients and p53 overexpression in the corresponding tumours was also observed. We did not find a significant correlation between levels of serum p53 antibodies and TP53 mutations in the corresponding tumours.

HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation.

HIPK2 (homeodomain-interacting protein kinase-2) binds to and phosphorylates, at Ser and Thr residues, a large number of targets involved in cell division and cell fate decision in response to different physiological or stress stimuli. Inactivation of HIPK2 has been observed in human and mouse cancers supporting its role as a tumor suppressor. Despite the biological relevance of this kinase, very little is known on how HIPK2 becomes catalytically active. Based on sequence homologies, HIPK2 has been taxonomically classified as a subfamily member of the dual-specificity tyrosine-regulated kinases (DYRKs) and the activation-loop Y354 of HIPK2 has been found phosphorylated in different cells; however, the relevance of this Y phosphorylation is presently unknown. Here, we show that HIPK2, which is extensively phosphorylated at S/T sites throughout its functional domains, becomes catalytically active by autophosphorylation at the activation-loop Y354. In particular, we found that, in analogy to DYRKs, HIPK2-Y354 phosphorylation is an autocatalytic event and its prevention, through Y354 substitution with non-phosphorylatable amino acids or by using the kinase inhibitor purvalanol A, induces a strong reduction of the HIPK2 S/T-kinase activity on different substrates. Interestingly, at variance from DYRKs, inhibition of HIPK2-Y354 phosphorylation induces a strong out-of-target Y-kinase activity in cis and a strong cytoplasmic relocalization of the kinase. Together, these results demonstrate that the catalytic activity, substrate specificity, and subcellular localization of HIPK2 are regulated by autophosphorylation of its activation-loop Y354.

Che-1 phosphorylation by ATM/ATR and Chk2 kinases activates p53 transcription and the G2/M checkpoint.

Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.

NRF2 activation in BON­1 neuroendocrine cancer cells reduces the cytotoxic effects of a novel Ruthenium(II)­curcumin compound: A pilot study.

Gastroenteropancreatic neuroendocrine neoplasms (GEP­NEN) are a group of rare tumors whose specific pathogenetic mechanisms of resistance to therapies have not been completely revealed yet. Chemotherapy is the main therapeutic approach in patients with GEP­NEN, however, novel combination regimens and targeted therapy are continuously explored. In the present study, the anticancer effect of a novel Ruthenium (Ru)(II)­Bisdemethoxycurcumin (Ru­bdcurc) compound was evaluated in BON­1 cell line, one of the few cell lines derived from GEP­NEN, largely used in experimental research of this type of tumors. The experimental data revealed that the Ru­bdcurc compound induced cell death in a dose­dependent manner, in vitro. Biochemical studies demonstrated that, in response to the lower dose of treatment, BON­1 cells activated the nuclear factor erythroid 2­related factor 2 (NRF2) pathway with induction of some of its targets including catalase and p62 as well as of the antiapoptotic marker Bcl2, all acting as chemoresistance mechanisms. NRF2 induction associated also with increased expression of endogenous p53 which is reported to be dysfunctional in BON­1 cells and to inhibit apoptosis. Genetic or pharmacologic targeting of NRF2 inhibited the activation of the NRF2 pathway, as well as of endogenous dysfunctional p53, in response to the lower dose of Ru­bdcurc, increasing the cell death. To assess the interplay between NRF2 and dysfunctional p53, genetic targeting of p53 showed reduced activation of the NRF2 pathway in response to the lower dose of Ru­bdcurc, increasing the cell death. These findings identified for the first time a possible dysfunctional p53/NRF2 interplay in BON­1 cell line that can be a novel key determinant in cell resistance to cytotoxic agents to be evaluated also in GEP­NEN.

AurkA/TPX2 co-overexpression in nontransformed cells promotes genome instability through induction of chromosome mis-segregation and attenuation of the p53 signalling pathway.

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.

The synergism of SMC1A cohesin gene silencing and bevacizumab against colorectal cancer.

BACKGROUND: SMC1A is a subunit of the cohesin complex that participates in many DNA- and chromosome-related biological processes. Previous studies have established that SMC1A is involved in cancer development and in particular, is overexpressed in chromosomally unstable human colorectal cancer (CRC). This study aimed to investigate whether SMC1A could serve as a therapeutic target for CRC. METHODS: At first, we studied the effects of either SMC1A overexpression or knockdown in vitro. Next, the outcome of SMC1A knocking down (alone or in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor) was analyzed in vivo. RESULTS: We found that SMC1A knockdown affects cell proliferation and reduces the ability to grow in anchorage-independent manner. Next, we demonstrated that the silencing of SMC1A and the combo treatment were effective in increasing overall survival in a xenograft mouse model. Functional analyses indicated that both treatments lead to atypical mitotic figures and gene expression dysregulation. Differentially expressed genes were implicated in several pathways including gene transcription regulation, cellular proliferation, and other transformation-associated processes. CONCLUSIONS: These results indicate that SMC1A silencing, in combination with bevacizumab, can represent a promising therapeutic strategy for human CRC.

MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway.

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.

Serum p53 antibody detection in patients with impaired lung function.

BACKGROUND: TP53 gene mutations can lead to the expression of a dysfunctional protein that in turn may enable genetically unstable cells to survive and change into malignant cells. Mutant p53 accumulates early in cells and can precociously induce circulating anti-p53 antibodies (p53Abs); in fact, p53 overexpression has been observed in pre-neoplastic lesions, such as bronchial dysplasia, and p53Abs have been found in patients with chronic obstructive pulmonary disease, before the diagnosis of lung and other tobacco-related tumors. METHODS: A large prospective study was carried out, enrolling non-smokers, ex-smokers and smokers with or without the impairment of lung function, to analyze the incidence of serum p53Abs and the correlation with clinicopathologic features, in particular smoking habits and impairment of lung function, in order to investigate their possible role as early markers of the onset of lung cancer or other cancers. The p53Ab levels were evaluated by a specific ELISA in 675 subjects. RESULTS: Data showed that significant levels of serum p53Abs were present in 35 subjects (5.2%); no difference was observed in the presence of p53Abs with regard to age and gender, while p53Abs correlated with the number of cigarettes smoked per day and packs-year. Furthermore, serum p53Abs were associated with the worst lung function impairment. The median p53Ab level in positive subjects was 3.5 units/ml (range 1.2 to 65.3 units/ml). Only fifteen positive subjects participated in the follow-up, again resulting positive for serum p53Abs, and no evidence of cancer was found in these patients. CONCLUSION: The presence of serum p53Abs was found to be associated with smoking level and lung function impairment, both risk factors of cancer development. However, in our study we have not observed the occurrence of lung cancer or other cancers in the follow-up of positive subjects, therefore we cannot directly correlate the presence of serum p53Abs with cancer risk.

Mild Neurological Phenotype Associated with Hypomorphic Variants in the Ataxia-Telangiectasia Mutated Gene.

Background: Ataxia-telangiectasia (A-T) is a progressive multisystemic neurodegenerative disease. The phenotypic spectrum includes conditions (variant A-T) with mild, late-onset, and atypical clinical presentations characterized by the prevalence of dyskinetic rather than ataxic features. Cases: We describe the clinical presentations of 3 siblings with early-onset truncal ataxia without obvious neurological deterioration or biological markers of classic A-T phenotype. We performed functional and genetic evaluation of 3 siblings with very mild neurological phenotype. Genetic evaluation with a next-generation sequencing panel for genes causative of cerebellar ataxia detected 2 known ATM gene variants, missense c.9023G>A p.(Arg3008His), and leaky splicing c.1066-6T>G variants. Functional studies showed mildly reduced ATM expression and residual kinase activity in the probands compared with healthy controls. Conclusions: These results suggest the importance of investigating ATM variants even in the presence of clinical and biological atypical cases to ensure specific therapeutic regimens and oncological surveillance in these patients.

Homeodomain-interacting protein kinase-2 stabilizes p27(kip1) by its phosphorylation at serine 10 and contributes to cell motility.

HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb.

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.

Cross-Resistance Among Sequential Cancer Therapeutics: An Emerging Issue.

Over the past two decades, cancer treatment has benefited from having a significant increase in the number of targeted drugs approved by the United States Food and Drug Administration. With the introduction of targeted therapy, a great shift towards a new era has taken place that is characterized by reduced cytotoxicity and improved clinical outcomes compared to traditional chemotherapeutic drugs. At present, targeted therapies and other systemic anti-cancer therapies available (immunotherapy, cytotoxic, endocrine therapies and others) are used alone or in combination in different settings (neoadjuvant, adjuvant, and metastatic). As a result, it is not uncommon for patients affected by an advanced malignancy to receive subsequent anti-cancer therapies. In this challenging complexity of cancer treatment, the clinical pathways of real-life patients are often not as direct as predicted by standard guidelines and clinical trials, and cross-resistance among sequential anti-cancer therapies represents an emerging issue. In this review, we summarize the main cross-resistance events described in the diverse tumor types and provide insight into the molecular mechanisms involved in this process. We also discuss the current challenges and provide perspectives for the research and development of strategies to overcome cross-resistance and proceed towards a personalized approach.

Corrigendum: Cross-resistance among sequential cancer therapeutics: An emerging issue.

[This corrects the article DOI: 10.3389/fonc.2022.877380.].

Factors to Consider for the Correct Use of γH2AX in the Evaluation of DNA Double-Strand Breaks Damage Caused by Ionizing Radiation.

People exposed to ionizing radiation (IR) both for diagnostic and therapeutic purposes is constantly increasing. Since the use of IR involves a risk of harmful effects, such as the DNA DSB induction, an accurate determination of this induced DNA damage and a correct evaluation of the risk-benefit ratio in the clinical field are of key relevance. γH2AX (the phosphorylated form of the histone variant H2AX) is a very early marker of DSBs that can be induced both in physiological conditions, such as in the absence of specific external agents, and by external factors such as smoking, heat, background environmental radiation, and drugs. All these internal and external conditions result in a basal level of γH2AX which must be considered for the correct assessment of the DSBs after IR exposure. In this review we analyze the most common conditions that induce H2AX phosphorylation, including specific exogenous stimuli, cellular states, basic environmental factors, and lifestyles. Moreover, we discuss the most widely used methods for γH2AX determination and describe the principal applications of γH2AX scoring, paying particular attention to clinical studies. This knowledge will help us optimize the use of available methods in order to discern the specific γH2AX following IR-induced DSBs from the basal level of γH2AX in the cells.

Combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP.

BACKGROUND: Lamins, key nuclear lamina components, have been proposed as candidate risk biomarkers in different types of cancer but their accuracy is still debated. AKTIP is a telomeric protein with the property of being enriched at the nuclear lamina. AKTIP has similarity with the tumor susceptibility gene TSG101. AKTIP deficiency generates genome instability and, in p53-/- mice, the reduction of the mouse counterpart of AKTIP induces the exacerbation of lymphomas. Here, we asked whether the distribution of AKTIP is altered in cancer cells and whether this is associated with alterations of lamins. METHODS: We performed super-resolution imaging, quantification of lamin expression and nuclear morphology on HeLa, MCF7, and A549 tumor cells, and on non-transformed fibroblasts from healthy donor and HGPS (LMNA c.1824C > T p.Gly608Gly) and EDMD2 (LMNA c.775 T > G) patients. As proof of principle model combining a defined lamin alteration with a tumor cell setting, we produced HeLa cells exogenously expressing the HGPS lamin mutant progerin that alters nuclear morphology. RESULTS: In HeLa cells, AKTIP locates at less than 0.5 µm from the nuclear rim and co-localizes with lamin A/C. As compared to HeLa, there is a reduced co-localization of AKTIP with lamin A/C in both MCF7 and A549. Additionally, MCF7 display lower amounts of AKTIP at the rim. The analyses in non-transformed fibroblasts show that AKTIP mislocalizes in HGPS cells but not in EDMD2. The integrated analysis of lamin expression, nuclear morphology, and AKTIP topology shows that positioning of AKTIP is influenced not only by lamin expression, but also by nuclear morphology. This conclusion is validated by progerin-expressing HeLa cells in which nuclei are morphologically altered and AKTIP is mislocalized. CONCLUSIONS: Our data show that the combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP. The results also point to the fact that lamin alterations per se are not predictive of AKTIP mislocalization, in both non-transformed and tumor cells. In more general terms, this study supports the thesis that a combined analytical approach should be preferred to predict lamin-associated changes in tumor cells. This paves the way of next translational evaluation to validate the use of this combined analytical approach as risk biomarker.