Mutant p53 drives invasion by promoting integrin recycling.

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.

EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7.

EML4-ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.

Green-Light Activatable BODIPY and Coumarin 5'-Caps for Oligonucleotide Photocaging.

We synthesized two green-light activatable 5'-caps for oligonucleotides based on the BODIPY and coumarin scaffold. Both bear an alkyne functionality allowing their use in numerous biological applications. They were successfully incorporated in oligonucleotides via solid-phase synthesis. Copper-catalyzed alkyne-azide cycloaddition (CuAAC) using a bisazide photo-tether gave cyclic oligonucleotides that could be relinearized by activation with green light and were shown to exhibit high stability against exonucleases. Chemical ligation as another example for bioconjugation yielded oligonucleotides with an internal strand break site. Irradiation at 530 nm or 565 nm resulted in complete photolysis of both caging groups.

Interaction of p53 with the CCT complex promotes protein folding and wild-type p53 activity.

p53 is a transcription factor that mediates tumor suppressor responses. Correct folding of the p53 protein is essential for these activities, and point mutations that induce conformational instability of p53 are frequently found in cancers. These mutant p53s not only lose wild-type activity but can also acquire the ability to promote invasion and metastasis. We show that folding of wild-type p53 is promoted by an interaction with the chaperonin CCT. Depletion of this chaperone in cells results in the accumulation of misfolded p53, leading to a reduction in p53-dependent gene expression. Intriguingly, p53 proteins mutated to prevent the interaction with CCT show conformational instability and acquire an ability to promote invasion and random motility that is similar to the activity of tumor-derived p53 mutants. Our data therefore suggest that both growth suppression and cell invasion may be differentially regulated functions of wild-type p53.

Evaluation of the Role of p53 Tumour Suppressor Posttranslational Modifications and TTC5 Cofactor in Lung Cancer.

Mutations in the p53 tumor suppressor are found in over 50% of cancers. p53 function is controlled through posttranslational modifications and cofactor interactions. In this study, we investigated the posttranslationally modified p53, including p53 acetylated at lysine 382 (K382), p53 phosphorylated at serine 46 (S46), and the p53 cofactor TTC5/STRAP (Tetratricopeptide repeat domain 5/ Stress-responsive activator of p300-TTC5) proteins in lung cancer. Immunohistochemical (IHC) analysis of lung cancer tissues from 250 patients was carried out and the results were correlated with clinicopathological features. Significant associations between total or modified p53 with a higher grade of the tumour and shorter overall survival (OS) probability were detected, suggesting that mutant and/or modified p53 acts as an oncoprotein in these patients. Acetylated at K382 p53 was predominantly nuclear in some samples and cytoplasmic in others. The localization of the K382 acetylated p53 was significantly associated with the gender and grade of the disease. The TTC5 protein levels were significantly associated with the grade, tumor size, and node involvement in a complex manner. SIRT1 expression was evaluated in 50 lung cancer patients and significant positive correlation was found with p53 S46 intensity, whereas negative TTC5 staining was associated with SIRT1 expression. Furthermore, p53 protein levels showed positive association with poor OS, whereas TTC5 protein levels showed positive association with better OS outcome. Overall, our results indicate that an analysis of p53 modified versions together with TTC5 expression, upon testing on a larger sample size of patients, could serve as useful prognostic factors or drug targets for lung cancer treatment.

Controlling Coagulation in Blood with Red Light.

Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin-binding aptamer with a red-light photocleavable linker derived from Cy7 by Cu-catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood-restoring the blood's natural coagulation capability.

Biological relevance of cell-in-cell in cancers.

Cell-in-cell (CIC) is a term used to describe the presence of one, usually living, cell inside another cell that is typically considered non-phagocytic. Examples of this include tumour cells inside tumour cells (homotypic), mesenchymal stem cells inside tumour cells (heterotypic) or immune cells inside tumour cells (heterotypic). CIC formation can occur in cell lines and in tissues and it has been most frequently observed during inflammation and in cancers. Over the past 10 years, many researchers have studied CIC structures and a few different models have been proposed through which they can be formed, including entosis, cannibalism and emperipolesis among others. Recently, our laboratory discovered a role for mutant p53 in facilitating the formation of CIC and promoting genomic instability. These data and research by many others have uncovered a variety of molecules involved in CIC formation and have started to give us an idea of why they are formed and how they could contribute to oncogenic processes. In this perspective, we summarise current literature and speculate on the role of CIC in cancer biology.

Introducing LNAzo: More Rigidity for Improved Photocontrol of Oligonucleotide Hybridization.

Oligonucleotide-based therapeutics have made rapid progress in clinical treatment of a variety of disease indications. Since most therapeutic oligonucleotides serve more than just one function and tend to have a prolonged lifetime, spatio-temporal control of these functions would be desirable. Photoswitches like azobenzene have proven themselves as useful tools in this matter. Upon irradiation, the photoisomerization of the azobenzene moiety causes destabilization in adjacent base pairs, leading to a decreased hybridization affinity. Since the way the azobenzene is incorporated in the oligonucleotide is of utmost importance, we synthesized locked azobenzene C-nucleosides and compared their photocontrol capabilities to established azobenzene C-nucleosides in oligonucleotide test-sequences by means of fluorescence-, UV/Vis-, and CD-spectroscopy.

The Diverse Functions of Mutant 53, Its Family Members and Isoforms in Cancer.

The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53's inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics.

Perception of users on self-care of lower leg ulcers. / Percepção do usuário no autocuidado de úlcera em membros inferiores.

OBJECTIVE: To know the perception of users on self-care of ulcers in the lower limbs. METHOD: This is a qualitative, exploratory, and descriptive study conducted with 10 users registered in the complementary programme of a wound care clinic in Canoas, RS, Brazil. Data were collected using information from the medical records of the users and semi-structured interviews conducted between October and November 2016. RESULTS: The results were discussed according to thematic content analysis with three thematic categories: self-care and living with the ulcer, self-care deficit and supporting users with ulcer, and self-care and the ulcer care network, based on the Nursing Self-Care Deficit Theory. CONCLUSIONS: Self-care is the result of dialogue between the user/nurse/health workers and the link they establish when they share care. The support of family members and the service can facilitate or limit care.

Novel targets and interaction partners of mutant p53 Gain-Of-Function.

In many human cancers p53 expression is lost or a mutant p53 protein is expressed. Over the past 15 years it has become apparent that a large number of these mutant p53 proteins have lost wild type function, but more importantly have gained functions that promote tumorigenesis and drive chemo-resistance, invasion and metastasis. Many researchers have investigated the underlying mechanisms of these Gain-Of-Functions (GOFs) and it has become apparent that many of these functions are the result of mutant p53 hijacking other transcription factors. In this review, we summarize the latest research on p53 GOF and categorize these in light of the hallmarks of cancer as presented by Hannahan and Weinberg.

Mutant p53 regulates Dicer through p63-dependent and -independent mechanisms to promote an invasive phenotype.

The control and processing of microRNAs (miRs) is critical in the regulation of all cellular responses. Previous studies have suggested that a reduction in the expression of certain miRs, or an overall decrease in miR processing through the partial depletion of Dicer, can promote enhanced metastatic potential. We show here that Dicer depletion can promote the invasive behavior of cells that is reflected in enhanced recycling and activation of the growth factor receptors Met and EGF receptor. These responses are also seen in response to the expression of tumor-derived mutant p53s, and we show that mutant p53 can down-regulate Dicer expression through both direct inhibition of the TAp63-mediated transcriptional activation of Dicer and a TAp63-independent control of Dicer protein expression. Our results delineate a clear relationship between mutant p53, TAp63, and Dicer that might contribute to the metastatic function of mutant p53 but, interestingly, also reveal TAp63-independent functions of mutant p53 in controlling Dicer activity.

Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer.

p63 inhibits metastasis. Here, we show that p63 (both TAp63 and ΔNp63 isoforms) regulates expression of miR-205 in prostate cancer (PCa) cells, and miR-205 is essential for the inhibitory effects of p63 on markers of epithelial-mesenchymal transition (EMT), such as ZEB1 and vimentin. Correspondingly, the inhibitory effect of p63 on EMT markers and cell migration is reverted by anti-miR-205. p53 mutants inhibit expression of both p63 and miR-205, and the cell migration, in a cell line expressing endogenous mutated p53, can be abrogated by pre-miR-205 or silencing of mutated p53. In accordance with this in vitro data, ΔNp63 or miR-205 significantly inhibits the incidence of lung metastasis in vivo in a mouse tail vein model. Similarly, one or both components of the p63/miR-205 axis were absent in metastases or colonized lymph nodes in a set of 218 human prostate cancer samples. This was confirmed in an independent clinical data set of 281 patients. Loss of this axis was associated with higher Gleason scores, an increased likelihood of metastatic and infiltration events, and worse prognosis. These data suggest that p63/miR-205 may be a useful clinical predictor of metastatic behavior in prostate cancer.

Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion.

The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them--nardilysin (NRD1)--promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.

Modeling of Intracellular Taurine Levels Associated with Ovarian Cancer Reveals Activation of p53, ERK, mTOR and DNA-Damage-Sensing-Dependent Cell Protection.

Taurine, a non-proteogenic amino acid and commonly used nutritional supplement, can protect various tissues from degeneration associated with the action of the DNA-damaging chemotherapeutic agent cisplatin. Whether and how taurine protects human ovarian cancer (OC) cells from DNA damage caused by cisplatin is not well understood. We found that OC ascites-derived cells contained significantly more intracellular taurine than cell culture-modeled OC. In culture, elevation of intracellular taurine concentration to OC ascites-cell-associated levels suppressed proliferation of various OC cell lines and patient-derived organoids, reduced glycolysis, and induced cell protection from cisplatin. Taurine cell protection was associated with decreased DNA damage in response to cisplatin. A combination of RNA sequencing, reverse-phase protein arrays, live-cell microscopy, flow cytometry, and biochemical validation experiments provided evidence for taurine-mediated induction of mutant or wild-type p53 binding to DNA, activation of p53 effectors involved in negative regulation of the cell cycle (p21), and glycolysis (TIGAR). Paradoxically, taurine's suppression of cell proliferation was associated with activation of pro-mitogenic signal transduction including ERK, mTOR, and increased mRNA expression of major DNA damage-sensing molecules such as DNAPK, ATM and ATR. While inhibition of ERK or p53 did not interfere with taurine's ability to protect cells from cisplatin, suppression of mTOR with Torin2, a clinically relevant inhibitor that also targets DNAPK and ATM/ATR, broke taurine's cell protection. Our studies implicate that elevation of intracellular taurine could suppress cell growth and metabolism, and activate cell protective mechanisms involving mTOR and DNA damage-sensing signal transducti.

The nutritional supplement taurine activates p53-dependent and independent tumor suppressor mechanisms in various cellular models of ovarian cancer.

Loss of treatment-induced ovarian carcinoma (OC) growth suppression poses a major clinical challenge because it leads to disease recurrence. Therefore, there is a compelling need for well- -tolerated approaches that can support tumor growth-suppression after therapy is stopped. We have profiled ascites as OC tumor microenvironments to search for potential non-toxic soluble components that would activate tumor suppressor pathways in OC cells. Our investigations revealed that low levels of taurine, a non-proteogenic sulfonic amino acid, were present within OC ascites. Taurine supplementation, beyond levels found in ascites, induced growth suppression without causing cytotoxicity in various OC cells, including chemotherapy-resistant cell clones and patient-derived organoids representing primary or chemotherapy recovered disease. Inhibition of proliferation by taurine was linked to increased mutant or wild-type p53 proteins binding to DNA, induction of p21, and independently of p53, TIGAR expression. Taurine-induced activation of p21 and TIGAR was associated with suppression of cell-cycle progression, glycolysis, and mitochondrial respiration. Expression of p21 or TIGAR in OC cells mimicked taurine-induced growth suppression. Our studies support the potential therapeutic value of taurine supplementation in OC.

GOF Mutant p53 in Cancers: A Therapeutic Challenge.

TP53 is mutated in the majority of human cancers. Mutations can lead to loss of p53 expression or expression of mutant versions of the p53 protein. These mutant p53 proteins have oncogenic potential. They can inhibit any remaining WTp53 in a dominant negative manner, or they can acquire new functions that promote tumour growth, invasion, metastasis and chemoresistance. In this review we explore some of the mechanisms that make mutant p53 cells resistant to chemotherapy. As mutant p53 tumours are resistant to many traditional chemotherapies, many have sought to explore new ways of targeting mutant p53 tumours and reinstate chemosensitivity. These approaches include targeting of mutant p53 stability, mutant p53 binding partners and downstream pathways, p53 vaccines, restoration of WTp53 function, and WTp53 gene delivery. The current advances and challenges of these strategies are discussed.

Nuclear-cytosolic transport of COMMD1 regulates NF-kappaB and HIF-1 activity.

Copper metabolism MURR1 domain1 (COMMD1) is a novel inhibitor of the transcription factors NF-kappaB and HIF-1, which play important roles in inflammation and tumor growth, respectively. In this study, we identified two highly conserved nuclear export signals (NESs) in COMMD1 and revealed that these NESs were essential and sufficient to induce maximal nuclear export of COMMD1. Inhibition of CRM1-mediated nuclear export by Leptomycin B resulted in nuclear accumulation of COMMD1. In addition, low oxygen concentrations induced the active export of COMMD1 from the nucleus in a CRM1-dependent manner. Disruption of the NESs in COMMD1 increased the repression of COMMD1 in transcriptional activity of NF-kappaB and HIF-1. In conclusion, these data indicate that COMMD1 undergoes constitutive nucleocytoplasmic transport as a novel mechanism to regulate NF-kappaB and HIF-1 signaling.

Rab11-FIP1/RCP Functions as a Major Signalling Hub in the Oncogenic Roles of Mutant p53 in Cancer.

Rab11-FIP1 is a Rab effector protein that is involved in endosomal recycling and trafficking of various molecules throughout the endocytic compartments of the cell. The consequence of this can be increased secretion or increased membrane expression of those molecules. In general, expression of Rab11-FIP1 coincides with more tumourigenic and metastatic cell behaviour. Rab11-FIP1 can work in concert with oncogenes such as mutant p53, but has also been speculated to be an oncogene in its own right. In this perspective, we will discuss and speculate upon our observations that mutant p53 promotes Rab11-FIP1 function to not only promote invasive behaviour, but also chemoresistance by regulating a multitude of different proteins.

Mutant p53 promotes RCP-dependent chemoresistance coinciding with increased delivery of P-glycoprotein to the plasma membrane.

TP53 is the most frequently mutated gene in cancers. Mutations lead to loss of p53 expression or expression of a mutant protein. Mutant p53 proteins commonly lose wild-type function, but can also acquire novel functions in promoting metastasis and chemoresistance. Previously, we uncovered a role for Rab-coupling protein (RCP) in mutant p53-dependent invasion. RCP promotes endosomal recycling and signalling of integrins and receptor tyrosine kinases. In a screen to identify novel RCP-interacting proteins, we discovered P-glycoprotein (P-gp). Thus, we hypothesised that mutant p53 could promote chemoresistance through RCP-dependent recycling of P-gp. The interaction between RCP and P-gp was verified endogenously and loss of RCP or mutant p53 rendered cells more sensitive to cisplatin and etoposide. In mutant p53 cells we detected an RCP-dependent delivery of P-gp to the plasma membrane upon drug treatment and decreased retention of P-gp substrates. A co-localisation of P-gp and RCP was seen in mutant p53 cells, but not in p53-null cells upon chemotherapeutic exposure. In conclusion, mutant p53 expression enhanced co-localisation of P-gp and RCP to allow for rapid delivery of P-gp to the plasma membrane and increased resistance to chemotherapeutics.