Co-Expression of Multiple PAX Genes in Renal Cell Carcinoma (RCC) and Correlation of High PAX Expression with Favorable Clinical Outcome in RCC Patients.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, consisting of multiple distinct subtypes. RCC has the highest mortality rate amongst the urogenital cancers, with kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe carcinoma (KICH) being the most common subtypes. The Paired-box (PAX) gene family encodes transcription factors, which orchestrate multiple processes in cell lineage determination during embryonic development and organogenesis. Several PAX genes have been shown to be expressed in RCC following its onset and progression. Here, we performed real-time quantitative polymerase chain reaction (RT-qPCR) analysis on a series of human RCC cell lines, revealing significant co-expression of PAX2, PAX6, and PAX8. Knockdown of PAX2 or PAX8 mRNA expression using RNA interference (RNAi) in the A498 RCC cell line resulted in inhibition of cell proliferation, which aligns with our previous research, although no reduction in cell proliferation was observed using a PAX2 small interfering RNA (siRNA). We downloaded publicly available RNA-sequencing data and clinical histories of RCC patients from The Cancer Genome Atlas (TCGA) database. Based on the expression levels of PAX2, PAX6, and PAX8, RCC patients were categorized into two PAX expression subtypes, PAXClusterA and PAXClusterB, exhibiting significant differences in clinical characteristics. We found that the PAXClusterA expression subgroup was associated with favorable clinical outcomes and better overall survival. These findings provide novel insights into the association between PAX gene expression levels and clinical outcomes in RCC patients, potentially contributing to improved treatment strategies for RCC.

Phenotype Switching and the Melanoma Microenvironment; Impact on Immunotherapy and Drug Resistance.

Melanoma, a highly heterogeneous tumor, is comprised of a functionally diverse spectrum of cell phenotypes and subpopulations, including stromal cells in the tumor microenvironment (TME). Melanoma has been shown to dynamically shift between different transcriptional states or phenotypes. This is referred to as phenotype switching in melanoma, and it involves switching between quiescent and proliferative cell cycle states, and dramatic shifts in invasiveness, as well as changes in signaling pathways in the melanoma cells, and immune cell composition in the TME. Melanoma cell plasticity is associated with altered gene expression in immune cells and cancer-associated fibroblasts, as well as changes in extracellular matrix, which drive the metastatic cascade and therapeutic resistance. Therefore, resistance to therapy in melanoma is not only dependent on genetic evolution, but it has also been suggested to be driven by gene expression changes and adaptive phenotypic cell plasticity. This review discusses recent findings in melanoma phenotype switching, immunotherapy resistance, and the balancing of the homeostatic TME between the different melanoma cell subpopulations. We also discuss future perspectives of the biology of neural crest-like state(s) in melanoma.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body's immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

Can Immune Suppression and Epigenome Regulation in Placenta Offer Novel Insights into Cancer Immune Evasion and Immunotherapy Resistance?

Cancer is the second leading cause of mortality and morbidity in the developed world. Cancer progression involves genetic and epigenetic alterations, accompanied by aggressive changes, such as increased immune evasion, onset of metastasis, and drug resistance. Similar to cancer, DNA hypomethylation, immune suppression, and invasive cell behaviours are also observed in the human placenta. Mechanisms that lead to the acquisition of invasive behaviour, immune evasion, and drug and immunotherapy resistance are presently under intense investigations to improve patient outcomes. Here, we review current knowledge regarding the similarities between immune suppression and epigenome regulation, including the expression of repetitive elements (REs), endogenous retroviruses (ERVs) and transposable elements (TEs) in cells of the placenta and in cancer, which are associated with changes in immune regulation and invasiveness. We explore whether immune suppression and epigenome regulation in placenta offers novel insights into immunotherapy resistance in cancer, and we also discuss the implications and the knowledge gaps relevant to these findings, which are rapidly being accrued in these quite disparate research fields. Finally, we discuss potential linkages between TE, ERV and RE activation and expression, regarding mechanisms of immune regulation in placenta and cancer. A greater understanding of the role of immune suppression and associated epigenome regulation in placenta could help to elucidate some comparable mechanisms operating in cancer, and identify potential new therapeutic targets for treating cancer.

Differential Expression of BARD1 Isoforms in Melanoma.

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

Molecular endoscopic imaging for the detection of Barrett's metaplasia using biodegradable inorganic nanoparticles: An ex-vivo pilot study on human tissue.

BACKGROUND AND STUDY AIMS: Despite major technical advancements, endoscopic surveillance for detecting premalignant lesions in Barrett's esophagus is challenging because of their flat appearance with only subtle morphological changes. Molecular endoscopic imaging (MEI) using nanoparticles (NPs), coupled with fluorescently labeled antibody permits visualization of disease-specific molecular alterations. The aim of this ex vivo study was to assess the diagnostic applicability of MEI with NPs to detect Barrett's metaplasia. PATIENTS AND METHODS: Seven patients undergoing endoscopic surveillance of known Barrett's esophagus were recruited. Freshly resected biopsy specimens were incubated with NPs coupled with FITC labeled Muc-2 antibodies and examined with MEI. Fluorescence intensity from Barrett's mucosa and control specimens were compared, followed by histological confirmation. RESULTS: Fluorescence signals, indicating the presence of goblet cells, were noted for traditional MEI using Muc-2 antibodies in Barrett's intestinal metaplasia. Significantly stronger fluorescence signals were achieved with NPs coupled with FITC-conjugated Muc-2 antibodies. The results of MEI with NPs for the prediction of Barrett's metaplasia correlated with the final histopathological examination in all the cases. CONCLUSIONS: Highly-specific NPs detected Barrett's metaplasia more efficiently than conventional MEI in this first feasibility study. MEI was as effective as standard histopathology for identifying Muc-2 containing goblet cells for diagnosis of Barrett's metaplasia. (DRKS-ID: DRKS00017747).

Krebs Cycle Intermediate-Modified Carbonate Apatite Nanoparticles Drastically Reduce Mouse Tumor Burden and Toxicity by Restricting Broad Tissue Distribution of Anticancer Drugs.

The morphology, size, and surface area of nanoparticles (NPs), with the existence of functional groups on their surface, contribute to the drug binding affinity, distribution of the payload in different organs, and targeting of a particular tumor for exerting effective antitumor activity in vivo. However, the inherent chemical structure of NPs causing unpredictable biodistribution with a toxic outcome still poses a serious challenge in clinical chemotherapy. In this study, carbonate apatite (CA), citrate-modified CA (CMCA) NPs, and α-ketoglutaric acid-modified CA (α-KAMCA) NPs were employed as carriers of anticancer drugs for antitumor, pharmacokinetic, and toxicological analysis in a murine breast cancer model. The results demonstrated almost five-fold enhanced tumor regression in the cyclophosphamide (CYP)-loaded α-KAMCA NP-treated group compared to the group treated with CYP only. Also, NPs promoted much higher drug accumulation in blood and tumor in comparison with the drug injected without a carrier. In addition, doxorubicin (DOX)-loaded NPs exhibited less accumulation in the heart, indicating less potential myocardial toxicity in mice compared to free DOX. Our findings, thus, conclude that CA, CMCA, and α-KAMCA NPs extended the circulation half-life and enhanced the anticancer effect with reduced toxicity of conventional chemotherapeutics in healthy organs, signifying that they are promising drug delivery devices in breast cancer treatment.

α-Ketoglutaric Acid-Modified Carbonate Apatite Enhances Cellular Uptake and Cytotoxicity of a Raf- Kinase Inhibitor in Breast Cancer Cells through Inhibition of MAPK and PI-3 Kinase Pathways.

AZ628 is a hydrophobic Raf-kinase inhibitor (rapidly accelerated fibrosarcoma) currently in clinical trial of various cancer. The physicochemical properties of hydrophobic drugs that affect the drug-particle interactions and cause aggregation of drugs and particles might be the key aspect to impede effective drug delivery. Retaining smaller particle size is the prerequisite to overcome the opsonization and improve cytotoxicity in the targeted region. Carbonate apatite (CA), an attractive biodegradable vector, has been used to carry both hydrophilic and hydrophobic drugs and release the payloads inside the cells following endocytosis. We incorporated AZ628 into CA and also modified it with α-ketoglutaric acid (α-KA) for reducing particle growth kinetics and increasing total surface area to improve the delivery of AZ628 by enhancing cellular uptake by breast cancer cells. AZ628-loaded nanoparticles of CA and α-KA-modified CA (α-KAMCA) were synthesized and evaluated in MCF-7 and 4T1 cell lines by measuring cytotoxicity and cellular uptake analysis. HPLC (high-performance liquid chromatography) assay was performed to quantify the binding affinity of the nanocarriers towards the drug. Western blot analysis was done to see the activation and expression levels of Akt, MAPK (mitogen-activated protein kinase) pathways and Caspase-3. Zetasizer was used to measure the particle size along with the surface charge. α-KAMCA showed almost 88% encapsulation efficacy for AZ628 with around 21% enhanced cellular uptake of the drug in two different breast cancer cell lines. These findings suggest that α-KAMCA could be a promising therapeutic tool to carry AZ628 for breast cancer treatment.

Citrate Association Dramatically Reduces Diameter with Concomitant Increase in Uptake of Drug-Loaded Carbonate Apatite Particles in Breast Cancer Cells.

Inorganic nanoparticles are commonly employed as vectors for delivering drugs into cancer cells while decreasing undesired cytotoxicity in healthy tissues. Carbonate apatite is an attractive nonviral vector that releases drugs at acidic environment inside the cells following endocytosis. However, maintaining the smaller particle size is crucial for enhancing cellular uptake of drugs as well as prolonging their systemic circulation time. We aimed to modify carbonate apatite with citrate for reducing the growth kinetics of carbonate apatite particles and enhancing the cellular uptake of cyclophosphamide via endocytosis. Several concentrations of sodium citrate were used to fabricate citrate-modified carbonate apatite (CMCA) particle complexes in absence or presence of cyclophosphamide. The binding affinity of the drug towards the particles and its cellular uptake were measured by high-performance liquid chromatography (HPLC). The nanoparticles' average size and zeta potential were determined by Malvern Zetasizer. Fourier-transform infrared spectroscopy (FTIR) was performed to justify association of citrate with carbonate apatite. MTT assay was performed to evaluate the cell viability. CMCA exhibited 6% more binding efficiency for cyclophosphamide and promoted fast cellular uptake of cyclophosphamide with enhanced cytotoxicity in MCF-7 cells, compared to unmodified carbonate apatite. Therefore, CMCA nanoparticles have a high potential for intracellular delivery of anti-cancer drugs and demand for further investigated in animal models of cancer.

Duloxetine in Painful Diabetic Neuropathy: A Systematic Review.

OBJECTIVE: To systematically review the evidence for duloxetine in the management of painful diabetic neuropathy (PDN). METHODS: Electronic searches of Medline and PubMed were performed from 2005 till October 2015 using medical subject headings and free-text words. Two independent reviewers extracted the data and assessed the methodological quality of the selected studies. RESULTS: Twenty-three studies met our inclusion criteria and 8 were considered of high quality and were included to this review. Because of heterogeneity of the studies included in this review, statistical pooling of the data was not possible. We found good evidence for use of duloxetine in PDN over placebo and pregabalin but there was no benefit of duloxetine over amitriptyline. CONCLUSIONS: Duloxetine has a beneficial effect over placebo. Nevertheless, the evidence of superiority of duloxetine over pregabalin and amitriptyline should be explored further as there was only 1 trial for each category. Provided majority of the PDN patients share cardiovascular complications, use of duloxetine will be a good option for treating pain associated with PDN over amitriptyline. Future randomized controlled trials should be designed keeping this in mind.