A Review of CAR-T Combination Therapies for Treatment of Gynecological Cancers.

CAR-T cell therapy offers a promising way for prolonged cancer remission, specifically in the case of blood cancers. However, its application in the treatment of solid tumors still faces many limitations. This review paper provides a comprehensive overview of the challenges and strategies associated with CAR-T cell therapy for solid tumors, with a focus on gynecological cancer. This study discusses the limitations of CAR-T therapy for solid tumor treatment, such as T cell exhaustion, stromal barrier, and antigen shedding. Additionally, it addresses possible approaches to increase CAR-T efficacy in solid tumors, including combination therapies with checkpoint inhibitors and chemotherapy, as well as the novel approach of combining CAR-T with oncolytic virotherapy. Given the lack of comprehensive research on CAR-T combination therapies for treating gynecological cancers, this review aims to provide insights into the current landscape of combination therapies for solid tumors and highlight the potential of such an approach in gynecology.

The Role of p53 in Nanoparticle-Based Therapy for Cancer.

p53 is arguably one of the most important tumor suppressor genes in humans. Due to the paramount importance of p53 in the onset of cell cycle arrest and apoptosis, the p53 gene is found either silenced or mutated in the vast majority of cancers. Furthermore, activated wild-type p53 exhibits a strong bystander effect, thereby activating apoptosis in surrounding cells without being physically present there. For these reasons, p53-targeted therapy that is designed to restore the function of wild-type p53 in cancer cells seems to be a very appealing therapeutic approach. Systemic delivery of p53-coding DNA or RNA using nanoparticles proved to be feasible both in vitro and in vivo. In fact, one p53-based therapeutic (gendicine) is currently approved for commercial use in China. However, the broad use of p53-based therapy in p53-inactivated cancers is severely restricted by its inadequate efficacy. This review highlights the current state-of-the-art in this area of biomedical research and also discusses novel approaches that may help overcome the shortcomings of p53-targeting nanomedicine.

Non-Immunoglobulin Synthetic Binding Proteins for Oncology.

Extensive application of technologies like phage display in screening peptide and protein combinatorial libraries has not only facilitated creation of new recombinant antibodies but has also significantly enriched repertoire of the protein binders that have polypeptide scaffolds without homology to immunoglobulins. These innovative synthetic binding protein (SBP) platforms have grown in number and now encompass monobodies/adnectins, DARPins, lipocalins/anticalins, and a variety of miniproteins such as affibodies and knottins, among others. They serve as versatile modules for developing complex affinity tools that hold promise in both diagnostic and therapeutic settings. An optimal scaffold typically has low molecular weight, minimal immunogenicity, and demonstrates resistance against various challenging conditions, including proteolysis - making it potentially suitable for peroral administration. Retaining functionality under reducing intracellular milieu is also advantageous. However, paramount to its functionality is the scaffold's ability to tolerate mutations across numerous positions, allowing for the formation of a sufficiently large target binding region. This is achieved through the library construction, screening, and subsequent expression in an appropriate system. Scaffolds that exhibit high thermodynamic stability are especially coveted by the developers of new SBPs. These are steadily making their way into clinical settings, notably as antagonists of oncoproteins in signaling pathways. This review surveys the diverse landscape of SBPs, placing particular emphasis on the inhibitors targeting the oncoprotein KRAS, and highlights groundbreaking opportunities for SBPs in oncology.

Artificial genetic polymers against human pathologies.

Originally discovered by Nielsen in 1991, peptide nucleic acids and other artificial genetic polymers have gained a lot of interest from the scientific community. Due to their unique biophysical features these artificial hybrid polymers are now being employed in various areas of theranostics (therapy and diagnostics). The current review provides an overview of their structure, principles of rational design, and biophysical features as well as highlights the areas of their successful implementation in biology and biomedicine. Finally, the review discusses the areas of improvement that would allow their use as a new class of therapeutics in the future.

Alpha-actnin-4 (ACTN4) selectively affects the DNA double-strand breaks repair in non-small lung carcinoma cells.

BACKGROUND: ACTN4 is an actin-binding protein involved in many cellular processes, including cancer development. High ACTN4 expression is often associated with a poor prognosis. However, it has been identified as a positive marker for platinum-based adjuvant chemotherapy for non-small cell lung cancer (NSCLC). The goal of our study was to investigate the involvement of ACTN4 in the NSCLC cells' response to the genotoxic drugs. RESULTS: We generated H1299 cells with the ACTN4 gene knock-out (ACTN4 KO), using the CRISPR/Cas9 system. The resistance of the cells to the cisplatin and etoposide was analyzed with the MTT assay. We were also able to estimate the efficiency of DNA repair through the DNA comet assay and gamma-H2AX staining. Possible ACTN4 effects on the non-homologous end joining (NHEJ) and homologous recombination (HR) were investigated using pathway-specific reporter plasmids and through the immunostaining of the key proteins. We found that the H1299 cells with the ACTN4 gene knock-out did not show cisplatin-resistance, but did display a higher resistance to the topoisomerase II inhibitors etoposide and doxorubicin, suggesting that ACTN4 might be somehow involved in the repair of DNA strand breaks. Indeed, the H1299 ACTN4 KO cells repaired etoposide- and doxorubicin-induced DNA breaks more effectively than the control cells. Moreover, the ACTN4 gene knock-out enhanced NHEJ and suppressed HR efficiency. Supporting the data, the depletion of ACTN4 resulted in the faster assembly of the 53BP1 foci with a lower number of the phospho-BRCA1 foci after the etoposide treatment. CONCLUSIONS: Thus, we are the first to demonstrate that ACTN4 may influence the resistance of cancer cells to the topoisomerase II inhibitors, and affect the efficiency of the DNA double strand breaks repair. We hypothesize that ACTN4 interferes with the assembly of the NHEJ and HR complexes, and hence regulates balance between these DNA repair pathways.

How Should the Worldwide Knowledge of Traditional Cancer Healing Be Integrated with Herbs and Mushrooms into Modern Molecular Pharmacology?

Traditional herbal medicine (THM) is a "core" from which modern medicine has evolved over time. Besides this, one third of people worldwide have no access to modern medicine and rely only on traditional medicine. To date, drugs of plant origin, or their derivates (paclitaxel, vinblastine, vincristine, vinorelbine, etoposide, camptothecin, topotecan, irinotecan, and omacetaxine), are very important in the therapy of malignancies and they are included in most chemotherapeutic regimes. To date, 391,000 plant and 14,000 mushroom species exist. Their medical and biochemical capabilities have not been studied in detail. In this review, we systematized the information about plants and mushrooms, as well as their active compounds with antitumor properties. Plants and mushrooms are divided based on the regions where they are used in ethnomedicine to treat malignancies. The majority of their active compounds with antineoplastic properties and mechanisms of action are described. Furthermore, on the basis of the available information, we divided them into two priority groups for research and for their potential of use in antitumor therapy. As there are many prerequisites and some examples how THM helps and strengthens modern medicine, finally, we discuss the positive points of THM and the management required to transform and integrate THM into the modern medicine practice.

Zeb1-mediated autophagy enhances resistance of breast cancer cells to genotoxic drugs.

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.

Dual Role of p73 in Cancer Microenvironment and DNA Damage Response.

Understanding the mechanisms that regulate cancer progression is pivotal for the development of new therapies. Although p53 is mutated in half of human cancers, its family member p73 is not. At the same time, isoforms of p73 are often overexpressed in cancers and p73 can overtake many p53 functions to kill abnormal cells. According to the latest studies, while p73 represses epithelial-mesenchymal transition and metastasis, it can also promote tumour growth by modulating crosstalk between cancer and immune cells in the tumor microenvironment, M2 macrophage polarisation, Th2 T-cell differentiation, and angiogenesis. Thus, p73 likely plays a dual role as a tumor suppressor by regulating apoptosis in response to genotoxic stress or as an oncoprotein by promoting the immunosuppressive environment and immune cell differentiation.

The p53 family member p73 in the regulation of cell stress response.

During oncogenesis, cells become unrestrictedly proliferative thereby altering the tissue homeostasis and resulting in subsequent hyperplasia. This process is paralleled by resumption of cell cycle, aberrant DNA repair and blunting the apoptotic program in response to DNA damage. In most human cancers these processes are associated with malfunctioning of tumor suppressor p53. Intriguingly, in some cases two other members of the p53 family of proteins, transcription factors p63 and p73, can compensate for loss of p53. Although both p63 and p73 can bind the same DNA sequences as p53 and their transcriptionally active isoforms are able to regulate the expression of p53-dependent genes, the strongest overlap with p53 functions was detected for p73. Surprisingly, unlike p53, the p73 is rarely lost or mutated in cancers. On the contrary, its inactive isoforms are often overexpressed in cancer. In this review, we discuss several lines of evidence that cancer cells develop various mechanisms to repress p73-mediated cell death. Moreover, p73 isoforms may promote cancer growth by enhancing an anti-oxidative response, the Warburg effect and by repressing senescence. Thus, we speculate that the role of p73 in tumorigenesis can be ambivalent and hence, requires new therapeutic strategies that would specifically repress the oncogenic functions of p73, while keeping its tumor suppressive properties intact.

Regulation of autophagy flux by E3 ubiquitin ligase Pirh2 in lung cancer.

Autophagy is a special catabolic cellular program that is induced in response to deprivation of nutrients and energy starvation. During the execution of this program, cellular components, including aggregates, as well as damaged organelles and some proteins are encapsulated in special vesicles known as autophagosomes and subsequently are degraded after fusion of autophagosomes with lysosomes. Importantly, at late stages of tumorigenesis cancer cells employ autophagy to sustain proliferation in unfavorable conditions, including anti-cancer drug therapy. E3 ubiquitin ligases play an important role in controlling autophagy. Here we demonstrate that the E3 ligase, a p53-induced RING-H2 protein (Pirh2), is involved in the regulation of autophagy in non-small cell lung cancer cells. Knockdown of Pirh2 decreased the expression of genes involved in all steps of autophagy. Concomitantly, Pirh2 knockdown cell lines exhibited much less of the processed form of LC3 compared to the respective cell lines with normal levels of Pirh2. These results were confirmed by the immune fluorescence microscopy using LC3 antibody and the LysoTracker dye. In agreement with the protective role of autophagy, cells with attenuated expression of Pirh2 were more sensitive to the treatment with doxorubicin. Collectively, we have uncovered a novel function of Pirh2 in the regulation of autophagy in lung cancer cells.

Emerging roles of cancer-testis antigenes, semenogelin 1 and 2, in neoplastic cells.

Cancer-testicular Antigens (CTAs) belong to a group of proteins that under normal conditions are strictly expressed in a male's reproductive tissues. However, upon malignisation, they are frequently re-expressed in neoplastic tissues of various origin. A number of studies have shown that different CTAs affect growth, migration and invasion of tumor cells and favor cancer development and metastasis. Two members of the CTA group, Semenogelin 1 and 2 (SEMG1 and SEMG2, or SEMGs) represent the major component of human seminal fluid. They regulate the motility and capacitation of sperm. They are often re-expressed in different malignancies including breast cancer. However, there is almost no information about the functional properties of SEMGs in cancer cells. In this review, we highlight the role of SEMGs in the reproductive system and also summarize the data on their expression and functions in malignant cells of various origins.

SEMG1/2 augment energy metabolism of tumor cells.

SEMG1 and SEMG2 genes belong to the family of cancer-testis antigens (CTAs), whose expression normally is restricted to male germ cells but is often restored in various malignancies. High levels of SEMG1 and SEMG2 expression are detected in prostate, renal, and lung cancer as well as hemoblastosis. However, the functional importance of both SEMGs proteins in human neoplasms is still largely unknown. In this study, by using a combination of the bioinformatics and various cellular and molecular assays, we have demonstrated that SEMG1 and SEMG2 are frequently expressed in lung cancer clinical samples and cancer cell lines of different origins and are negatively associated with the survival rate of cancer patients. Using the pull-down assay followed by LC-MS/MS mass-spectrometry, we have identified 119 proteins associated with SEMG1 and SEMG2. Among the SEMGs interacting proteins we noticed two critical glycolytic enzymes-pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Importantly, we showed that SEMGs increased the protein level and activity of both PKM2 and LDHA. Further, both SEMGs increased the membrane mitochondrial potential (MMP), glycolysis, respiration, and ROS production in several cancer cell lines. Taken together, these data provide first evidence that SEMGs can up-regulate the energy metabolism of cancer cells, exemplifying their oncogenic features.

Attenuation of p53 mutant as an approach for treatment Her2-positive cancer.

Breast cancer is one of the world's leading causes of oncological disease-related death. It is characterized by a high degree of heterogeneity on the clinical, morphological, and molecular levels. Based on molecular profiling breast carcinomas are divided into several subtypes depending on the expression of a number of cell surface receptors, e.g., ER, PR, and HER2. The Her2-positive subtype occurs in ~10-15% of all cases of breast cancer, and is characterized by a worse prognosis of patient survival. This is due to a high and early relapse rate, as well as an increased level of metastases. Several FDA-approved drugs for the treatment of Her2-positive tumors have been developed, although eventually cancer cells develop drug resistance. These drugs target either the homo- or heterodimerization of Her2 receptors or the receptors' RTK activity, both of them being critical for the proliferation of cancer cells. Notably, Her2-positive cancers also frequently harbor mutations in the TP53 tumor suppressor gene, which exacerbates the unfavorable prognosis. In this review, we describe the molecular mechanisms of RTK-specific drugs and discuss new perspectives of combinatorial treatment of Her2-positive cancers through inhibition of the mutant form of p53.

Effects of Mycoplasmas on the Host Cell Signaling Pathways.

Mycoplasmas are the smallest free-living organisms. Reduced sizes of their genomes put constraints on the ability of these bacteria to live autonomously and make them highly dependent on the nutrients produced by host cells. Importantly, at the organism level, mycoplasmal infections may cause pathological changes to the host, including cancer and severe immunological reactions. At the molecular level, mycoplasmas often activate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) inflammatory response and concomitantly inhibit the p53-mediated response, which normally triggers the cell cycle and apoptosis. Thus, mycoplasmal infections may be considered as cancer-associated factors. At the same time, mycoplasmas through their membrane lipoproteins (LAMPs) along with lipoprotein derivatives (lipopeptide MALP-2, macrophage-activating lipopeptide-2) are able to modulate anti-inflammatory responses via nuclear translocation and activation of the Nrf2 (nuclear factor-E2-related anti-inflammatory transcription factor 2). Thus, interactions between mycoplasmas and host cells are multifaceted and depend on the cellular context. In this review, we summarize the current information on the role of mycoplasmas in affecting the host's intracellular signaling mediated by the interactions between transcriptional factors p53, Nrf2, and NF-κB. A better understanding of the mechanisms underlying pathologic processes associated with reprogramming eukaryotic cells that arise during the mycoplasma-host cell interaction should facilitate the development of new therapeutic approaches to treat oncogenic and inflammatory processes.

Role of ACTN4 in Tumorigenesis, Metastasis, and EMT.

The actin-binding protein ACTN4 belongs to a family of actin-binding proteins and is a non-muscle alpha-actinin that has long been associated with cancer development. Numerous clinical studies showed that changes in ACTN4 gene expression are correlated with aggressiveness, invasion, and metastasis in certain tumors. Amplification of the 19q chromosomal region where the gene is located has also been reported. Experimental manipulations with ACTN4 expression further confirmed its involvement in cell proliferation, motility, and epithelial-mesenchymal transition (EMT). However, both clinical and experimental data suggest that the effects of ACTN4 up- or down-regulation may vary a lot between different types of tumors. Functional studies demonstrated its engagement in a number of cytoplasmic and nuclear processes, ranging from cytoskeleton reorganization to regulation of different signaling pathways. Such a variety of functions may be the reason behind cell type and cell line specific responses. Herein, we will review research progress and controversies regarding the prognostic and functional significance of ACTN4 for tumorigenesis.

Autophagy suppresses the pathogenic immune response to dietary antigens in cystic fibrosis.

Under physiological conditions, a finely tuned system of cellular adaptation allows the intestinal mucosa to maintain the gut barrier function while avoiding excessive immune responses to non-self-antigens from dietary origin or from commensal microbes. This homeostatic function is compromised in cystic fibrosis (CF) due to loss-of-function mutations in the CF transmembrane conductance regulator (CFTR). Recently, we reported that mice bearing defective CFTR are abnormally susceptible to a celiac disease-like enteropathy, in thus far that oral challenge with the gluten derivative gliadin elicits an inflammatory response. However, the mechanisms through which CFTR malfunction drives such an exaggerated response to dietary protein remains elusive. Here we demonstrate that the proteostasis regulator/transglutaminase 2 (TGM2) inhibitor cysteamine restores reduced Beclin 1 (BECN1) protein levels in mice bearing cysteamine-rescuable F508del-CFTR mutant, either in homozygosis or in compound heterozygosis with a null allele, but not in knock-out CFTR mice. When cysteamine restored BECN1 expression, autophagy was increased and gliadin-induced inflammation was reduced. The beneficial effects of cysteamine on F508del-CFTR mice were lost when these mice were backcrossed into a Becn1 haploinsufficient/autophagy-deficient background. Conversely, the transfection-enforced expression of BECN1 in human intestinal epithelial Caco-2 cells mitigated the pro-inflammatory cellular stress response elicited by the gliadin-derived P31-43 peptide. In conclusion, our data provide the proof-of-concept that autophagy stimulation may mitigate the intestinal malfunction of CF patients.

Orphan receptor NR4A3 is a novel target of p53 that contributes to apoptosis.

Major tumor suppressor and transcription factor p53 coordinates expression of many genes hence affecting critical cellular functions including cell cycle, senescence, and apoptosis. The NR4A family of orphan receptors (NR4A1-3) belongs to the superfamily of nuclear receptors. They regulate genes involved in proliferation, cell migration, and apoptosis. In this study, we report an identification of NR4A3 as a direct transcriptional target of p53. Using various techniques, we showed that p53 directly bound the promoter of NR4A3 gene and induced its transcription. Functionally, over-expression of NR4A3 attenuated proliferation of cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes, PUMA and Bax. Knockdown of NR4A3 reversed these phenotypes. Importantly, NR4A3 exhibited tumor suppressive functions both in p53-dependent and independent manner. In addition, NR4A3 physically interacted with an anti-apoptotic Bcl-2 protein hence sequestering it from blunting apoptosis. These observations were corroborated by the bioinformatics analysis, which demonstrated a correlation between high levels of NR4A3 expression and better survival of breast and lung cancer patients. Collectively, our studies revealed a novel transcriptional target of p53, NR4A3, which triggers apoptosis and thus likely has a tumor suppressive role in breast and lung cancers.

TG2 regulates the heat-shock response by the post-translational modification of HSF1.

Heat-shock factor 1 (HSF1) is the master transcription factor that regulates the response to proteotoxic stress by controlling the transcription of many stress-responsive genes including the heat-shock proteins. Here, we show a novel molecular mechanism controlling the activation of HSF1. We demonstrate that transglutaminase type 2 (TG2), dependent on its protein disulphide isomerase activity, triggers the trimerization and activation of HSF1 regulating adaptation to stress and proteostasis impairment. In particular, we find that TG2 loss of function correlates with a defect in the nuclear translocation of HSF1 and in its DNA-binding ability to the HSP70 promoter. We show that the inhibition of TG2 restores the unbalance in HSF1-HSP70 pathway in cystic fibrosis (CF), a human disorder characterized by deregulation of proteostasis. The absence of TG2 leads to an increase of about 40% in CFTR function in a new experimental CF mouse model lacking TG2. Altogether, these results indicate that TG2 plays a key role in the regulation of cellular proteostasis under stressful cellular conditions through the modulation of the heat-shock response.

Co-expression of RelA/p65 and ACTN4 induces apoptosis in non-small lung carcinoma cells.

Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.

Extracellular Proteasomes Are Deficient in 19S Subunits as Revealed by iTRAQ Quantitative Proteomics.

Proteasome-mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well-documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty-two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP-PIPs were associated with protein biosynthesis and, unlike CP-PIPs, were under represented by chaperon/ATP-binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 232: 842-851, 2017. © 2016 Wiley Periodicals, Inc.