PIK3/Akt/mTOR pathway alterations in metastatic castration-sensitive prostate cancer.

BACKGROUND: Alterations in the PIK3/Akt/mTOR pathway are commonly seen in metastatic castration-sensitive prostate cancer (mCSPC), however their role in outcomes is unknown. We aim to evaluate the prognostic significance as well as the genetic landscape of PIK3/Akt/mTOR pathway alteration in mCSPC. METHODS: Fourhundred and seventy-two patients with mCSPC were included who underwent next generation sequencing. PIK3/Akt/mTor pathway alterations were defined as mutations in Akt1, mTOR, PIK3CA, PIK3CB, PIK3R1, PTEN, TSC1, and TSC2. Endpoints of interests were radiographic progression-free survival (rPFS), time to development of castration resistant prostate cancer (tdCRPC), and overall survival (OS). Kaplan-Meier analysis was performed and Cox regression hazard ratios (HR) were calculated. RESULTS: One hundred and fifty-two (31.9%) patients harbored a PIK3/Akt/mTOR pathway alteration. Median rPFS and tdCRPC were 23.7 and 21.0 months in PIK3/Akt/mTOR altered compared to 32.8 (p = 0.08) and 32.1 months (p = 0.002) in wildtype tumors. On multivariable analysis PIK3/Akt/mTOR pathway alterations were associated with tdCRPC (HR 1.43, 95% CI, 1.05-1.94, p = 0.02), but not rPFS [Hazard ratio (HR) 1.20, 95% confidence interval (CI), 0.90-1.60, p = 0.21]. PIK3/Akt/mTOR pathway alterations were more likely to be associated with concurrent mutations in TP53 (40% vs. 28%, p = 0.01) and TMPRSS2-ERG (37% vs. 26%, p = 0.02) than tumors without PIK3/Akt/mTOR pathway alterations. Concurrent mutations were typically associated with shorter median times to rPFS and tdCRPC. DAVID analysis showed p53 signaling and angiogenesis pathways were enriched in PIK3/Akt/mTOR pathway altered tumors while beta-catenin binding and altered BRCA pathway were enriched in PIK3/Akt/mTOR pathway wildtype tumors. CONCLUSIONS: PIK3/Akt/mTOR pathway alterations were common in mCSPC and associated with poorer prognosis. The genetic landscape of PIK3/Akt/mTOR pathway altered tumors differed from wildtype tumors. Additional studies are needed to better understand and target the PIK3/Akt/mTOR pathway in mCSPC.

Glioblastoma Stem Cell Targeting Peptide Isolated Through Phage Display Binds Cadherin 2.

Glioblastoma stem cells (GSCs) have unique properties of self-renewal and tumor initiation that make them potential therapeutic targets. Development of effective therapeutic strategies against GSCs requires both specificity of targeting and intracranial penetration through the blood-brain barrier. We have previously demonstrated the use of in vitro and in vivo phage display biopanning strategies to isolate glioblastoma targeting peptides. Here we selected a 7-amino acid peptide, AWEFYFP, which was independently isolated in both the in vitro and in vivo screens and demonstrated that it was able to target GSCs over differentiated glioma cells and non-neoplastic brain cells. When conjugated to Cyanine 5.5 and intravenously injected into mice with intracranially xenografted glioblastoma, the peptide localized to the site of the tumor, demonstrating intracranial tumor targeting specificity. Immunoprecipitation of the peptide with GSC proteins revealed Cadherin 2 as the glioblastoma cell surface receptor targeted by the peptides. Peptide targeting of Cadherin 2 on GSCs was confirmed through ELISA and in vitro binding analysis. Interrogation of glioblastoma databases demonstrated that Cadherin 2 expression correlated with tumor grade and survival. These results confirm that phage display can be used to isolate unique tumor-targeting peptides specific for glioblastoma. Furthermore, analysis of these cell specific peptides can lead to the discovery of cell specific receptor targets that may serve as the focus of future theragnostic tumor-homing modalities for the development of precision strategies for the treatment and diagnosis of glioblastomas.

Phage display targeting identifies EYA1 as a regulator of glioblastoma stem cell maintenance and proliferation.

Glioblastoma (GBM) ranks among the most lethal of human malignancies with GBM stem cells (GSCs) that contribute to tumor growth and therapeutic resistance. Identification and isolation of GSCs continue to be a challenge, as definitive methods to purify these cells for study or targeting are lacking. Here, we leveraged orthogonal in vitro and in vivo phage display biopanning strategies to isolate a single peptide with GSC-specific binding properties. In silico analysis of this peptide led to the isolation of EYA1 (Eyes Absent 1), a tyrosine phosphatase and transcriptional coactivator. Validating the phage discovery methods, EYA1 was preferentially expressed in GSCs compared to differentiated tumor progeny. MYC is a central mediator of GSC maintenance but has been resistant to direct targeting strategies. Based on correlation and colocalization of EYA1 and MYC, we interrogated a possible interaction, revealing binding of EYA1 to MYC and loss of MYC expression upon targeting EYA1. Supporting a functional role for EYA1, targeting EYA1 expression decreased GSC proliferation, migration, and self-renewal in vitro and tumor growth in vivo. Collectively, our results suggest that phage display can identify novel therapeutic targets in stem-like tumor cells and that an EYA1-MYC axis represents a potential therapeutic paradigm for GBM.

WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages.

BACKGROUND: Although the standard treatment for the patients with recurrent and metastatic prostate cancer (CaP) is androgen deprivation therapy, castration-resistant prostate cancer (CRPC) eventually emerges. Our previous report indicated that bone morphogenetic protein 6 (BMP6) induced CRPC via tumour-infiltrating macrophages. In a separate line of study, we have observed that the WNT5A/BMP6 loop in CaP bone metastasis mediates resistance to androgen deprivation in tissue culture. Simultaneously, we have reported that BMP6 induced castration resistance in CaP cells via tumour-infiltrating macrophages. Therefore, our present study aims to investigate the mechanism of WNT5A and its interaction with macrophages on CRPC. METHODS: Doxycycline inducible WNT5A overexpression prostate cancer cell line was used for detailed mechanical study. RESULTS: WNT5A was associated with increased expression of chemokine ligand 2 (CCL2) in the human CaP cell line, LNCaP. Mechanistically, this induction of CCL2 by WNT5A is likely to be mediated via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling pathway. Our in vivo experiments demonstrated that the overexpression of WNT5A in LNCaP cells promoted castration resistance. Conversely, this resistance was inhibited with the removal of macrophages via clodronate liposomes. When patient-derived CaP LuCaP xenografts were analysed, high levels of WNT5A were correlated with increased levels of CCL2 and BMP6. In addition, higher levels of CCL2 and BMP6 were more commonly observed in intra-femoral transplanted tumours as compared to subcutaneous-transplanted tumours in the patient-derived PCSD1 bone-niche model. CONCLUSIONS: These findings collectively suggest that WNT5A may be a key gene that induces CRPC in the bone niche by recruiting and regulating macrophages through CCL2 and BMP6, respectively.

Pathological Outcome following Radical Prostatectomy in Men with Prostate Specific Antigen Greater than 10 ng/ml and Histologically Favorable Risk Prostate Cancer.

PURPOSE: Active surveillance is now the treatment of choice in men with low risk prostate cancer. Although there is no consensus on which patients are eligible for active surveillance, prostate specific antigen above 10 ng/ml is generally excluded. In an attempt to determine the validity of using a prostate specific antigen cutoff of 10 ng/ml to counsel men considering active surveillance we analyzed a multi-institution database to determine the pathological outcome in men with prostate specific antigen greater than 10 ng/ml but histologically favorable risk prostate cancer. MATERIALS AND METHODS: We queried a prospectively maintained database of men with histologically favorable risk prostate cancer who underwent radical prostatectomy between 2003 and 2015. The cohort was categorized into 3 groups based on prostate specific antigen level, including low-less than 10 ng/ml, intermediate-10 or greater to less than 20 and high-20 or greater. Associations of prostate specific antigen group with adverse pathological and oncologic outcomes were analyzed. RESULTS: Of 2,125 patients 1,327 were categorized with histologically favorable risk disease. However on multivariate analyses the rates of up staging and upgrading were similar between the intermediate and low prostate specific antigen groups. In contrast compared to the intermediate prostate specific antigen group the high group had higher incidences of up staging (p = 0.02) and upgrading to 4 + 3 or greater disease (p = 0.046). Biochemical recurrence-free survival rates revealed no pairwise intergroup differences except between the low and high groups. CONCLUSIONS: Patients with preoperatively elevated prostate specific antigen between 10 and less than 20 ng/ml who otherwise had histologically favorable risk prostate cancer were not at higher risk for adverse pathological outcomes than men with prostate specific antigen less than 10 ng/ml.

Radiation therapy for triple-negative breast cancer: from molecular insights to clinical perspectives.

INTRODUCTION: Triple-negative breast cancer (TNBC) lacks three common receptors, making traditional treatments less effective. This review highlights the importance of radiotherapy and emerging therapeutic strategies to enhance treatment outcomes in TNBC. AREAS COVERED: We conducted a literature search on PubMed for publications from 2000 to 2023 to discuss the critical role of radiotherapy in managing TNBC, emphasizing its applications from locoregional control to improving survival rates. The review explores molecular mechanisms underlying TNBC's radiotherapy response, including DNA damage repair and apoptosis, with a focus on BRCA1/2 mutations and Poly (ADP-ribose) polymerase (PARP) inhibition. We summarize preclinical and clinical research on radiosensitization strategies, from gene-targeted therapies to immunotherapy combinations, and the impact of post-mastectomy radiation therapy on locoregional control. The potential of personalized treatment approaches, integrating molecular profiling, targeted radiosensitizers, and the synergistic effects of radiotherapy with immunotherapy, is also discussed. EXPERT OPINION: Future TNBC treatment strategies should focus on precision medicine, integrating immunotherapy, developing novel radiosensitizers, and targeting biological pathways to overcome radioresistance. The integration of radiomics and artificial intelligence offers promising avenues for enhancing treatment personalization and efficacy, aiming to improve patient outcomes in TNBC.

Use of phage display biopanning as a tool to design CAR-T cells against glioma stem cells.

Background: Glioblastoma (GBM) is both the most common and aggressive type of primary brain tumor, associated with high mortality rates and resistance to conventional therapy. Despite recent advancements in knowledge and molecular profiling, recurrence of GBM is nearly inevitable. This recurrence has been attributed to the presence of glioma stem cells (GSCs), a small fraction of cells resistant to standard-of-care treatments and capable of self-renewal and tumor initiation. Therefore, targeting these cancer stem cells will allow for the development of more effective therapeutic strategies against GBM. We have previously identified several 7-amino acid length peptides which specifically target GSCs through in vitro and in vivo phage display biopanning. Methods and results: We have combined two of these peptides to create a dual peptide construct (EV), and demonstrated its ability to bind GSCs in vitro and target intracranial GBM in mouse models. A peptide pull-down performed with peptide EV followed by mass spectrometry determined N-cadherin as the binding partner of the peptide, which was validated by enzyme-linked immunosorbent assay and surface plasmon resonance. To develop cytotoxic cellular products aimed at specifically targeting GSCs, chimeric antigen receptors (CARs) were engineered containing the peptide EV in place of the single-chain variable fragment (scFv) as the antigen-binding domain. EV CAR-transduced T cells demonstrated specific reactivity towards GSCs by production of interferon-gamma when exposed to GSCs, in addition to the induction of GSC-specific apoptosis as illustrated by Annexin-V staining. Conclusion: These results exemplify the use of phage display biopanning for the isolation of GSC-targeting peptides, and their potential application in the development of novel cytotoxic therapies for GBM.

CD44+ lung cancer stem cell-derived pericyte-like cells cause brain metastases through GPR124-enhanced trans-endothelial migration.

Brain metastasis of lung cancer causes high mortality, but the exact mechanisms underlying the metastasis remain unclear. Here we report that vascular pericytes derived from CD44+ lung cancer stem cells (CSCs) in lung adenocarcinoma (ADC) potently cause brain metastases through the G-protein-coupled receptor 124 (GPR124)-enhanced trans-endothelial migration (TEM). CD44+ CSCs in perivascular niches generate the majority of vascular pericytes in lung ADC. CSC-derived pericyte-like cells (Cd-pericytes) exhibit remarkable TEM capacity to effectively intravasate into the vessel lumina, survive in the circulation, extravasate into the brain parenchyma, and then de-differentiate into tumorigenic CSCs to form metastases. Cd-pericytes uniquely express GPR124 that activates Wnt7-ß-catenin signaling to enhance TEM capacity of Cd-pericytes for intravasation and extravasation, two critical steps during tumor metastasis. Furthermore, selective disruption of Cd-pericytes, GPR124, or the Wnt7-ß-catenin signaling markedly reduces brain and liver metastases of lung ADC. Our findings uncover an unappreciated cellular and molecular paradigm driving tumor metastasis.

Combination of Immunotherapy and Radiation Therapy in Gastrointestinal Cancers: An Appraisal of the Current Literature and Ongoing Research.

Oncological outcomes are improving in gastrointestinal cancer with advancements in systemic therapies, and there is notable potential in combining immunotherapy and radiation therapy (RT) to allow for further improvements. Various preclinical and early phase II studies have shown promising synergy with immunotherapy and RT in gastrointestinal cancer. A few recent phase III studies have shown improved survival with the addition of immunotherapy to standard treatment for gastrointestinal cancer. The timing, duration, sequencing, and integration with other anti-cancer treatments are still areas of ongoing research. We have reviewed the published and ongoing studies of the combinations of immunotherapy and RT in gastrointestinal cancers.

Gold nanoparticle-assisted delivery of small, highly structured RNA into the nuclei of human cells.

Previous studies have shown that functionalized gold nanoparticles (AuNPs) can be used as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNA to living systems. Here, we report the ability of functionalized AuNP to deliver RNA aptamers into the nuclei of human cells. An in vitro-synthesized RNA aptamer specific to the ß-catenin protein was delivered into the HepG2 human cell line more efficiently via functionalized AuNP than liposome-based delivery, and resulted in nearly complete inhibition of ß-catenin binding to the p50 subunit of NF-κB in the nucleus. This inhibition led to repression of NF-κB p50-dependent transcription of CRP. Also, the ß-catenin aptamer in the nucleus led to down-regulation of ß-catenin-mediated transcriptional activity through the TCF complex and resulted in decrease in the levels of cyclin D, and c-myc mRNA by ~47% and ~57%, respectively. In addition, we used functionalized AuNP to deliver another RNA aptamer targeted to the p50 subunit of NF-κB into the A549 human cell line, and this was sufficient to induce apoptosis of the cells. Our findings demonstrate that AuNP GDS can be used to deliver small, highly structured RNA aptamers into the nucleus of human cells where they modulate the activity of transactivators by interacting with target proteins.

Enhancement of biomass, lipids, and polyunsaturated fatty acid (PUFA) production in Nannochloropsis oceanica with a combination of single wavelength light emitting diodes (LEDs) and low temperature in a three-phase culture system.

A three-phase culture system combining blue (465 nm) light-emitting diode (LED) wavelength as the first phase, green (550 nm) as the second phase, and temperature stress as the third phase was applied to a Nannochloropsis oceanica culture in 14-L photobioreactors. Microalgal growth promotion parameters were optimized in the first phase, followed by green LED stress for lipid production in the second phase. Maximum biomass and lipid production values of 0.75 gdcw L-1 and 57.6% (w/w) were obtained at an aeration rate of 0.50 vvm, with a light intensity of 250 µmol m-2 s-1 and 24:0 h light/dark cycle. Culture temperatures of 15, 10 and 5 °C were applied in the third phase, where temperature stress induced the production of monounsaturated and polyunsaturated fatty acid synthesis in N. oceanica. The production of α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid increased by 52% (w/w), 96% (w/w), and 77% (w/w), respectively, at 5 °C in the third phase.

A pseudoknot improves selection efficiency in ribosome display.

The size and diversity of ribosome display libraries depends upon stability of the complex formed between the ribosome, mRNA and translated protein. To investigate if mRNA secondary structure improves stability of the complex, we tested a pseudoknot, originating from the genomic RNA of infectious bronchitis virus (IBV), a member of the positive-stranded coronavirus group. We used the previously-isolated anti-DNA scFv, 3D8, as a target protein. During in vitro translation in rabbit reticulocyte lysate, we observed that incorporation of the pseudoknot into the mRNA resulted in production of a translational intermediate that corresponded to the expected size for ribosomal arrest at the pseudoknot. Complexes containing the mRNA pseudoknot exhibited a higher efficiency of affinity selection than that those without, indicating that the pseudoknot improves stability of the mRNA-ribosome-antibody complex in a eukaryotic translation system. Thus, in order to improve the efficiency of selection, this relatively short pseudoknot sequence could be incorporated into ribosome display.

Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome.

Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.

Physical and Functional Differences in Youth Baseball Players With and Without Throwing-Related Pain.

BACKGROUND: Identifying risk factors that contribute to shoulder and elbow pain within youth baseball players is important for improving injury prevention and rehabilitation strategies. HYPOTHESIS: Differences will exist between youth baseball players with and without a history of upper extremity pain on measures related to growth, shoulder performance, and baseball exposure. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A total of 84 youth baseball players were divided into 2 groups based on self-reported history of throwing-related arm pain. Group differences for growth-related, shoulder performance, and baseball exposure variables were analyzed by use of parametric and nonparametric tests, as appropriate. Multivariate logistic regression was used to assess variables most predictive of pain. RESULTS: The group of athletes with pain (n = 16) were taller and heavier, played more baseball per year, and had greater pitching velocity. Athletes with pain also had greater loss of internal rotation range of motion and greater side-to-side asymmetry in humeral retrotorsion (HRT), attributable to lower degrees of HRT within the nondominant humerus. Multivariate analysis revealed that player height was most predictive of pain, with a 1-inch increase in height resulting in a 77% increased risk of pain. CONCLUSION: Vertical growth that accompanies adolescence increases the risk of experiencing throwing-related pain in youth baseball players. Players who are taller, particularly those with faster pitching velocities, are at the greatest risk for developing pain and should be more carefully monitored for resultant injury. The degree of nondominant HRT may have a relationship to the development of pain, but further research is required to better understand the implications of this observation.

Cinnamomum cassia bark produced by solid-state fermentation with Phellinus baumii has the potential to alleviate atopic dermatitis-related symptoms.

In order to evaluate whether the aqueous fraction of Cinnamomum cassia produced by solid-state fermentation with Phellinus baumii (afCc/Pb) inhibits atopic symptoms in vivo, its efficacy was evaluated in an animal model of 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis. Immune-related cells were quantified using hematoxylin and eosin staining, and phenotypic cytokines, enzymes and the expression of other proteins in the animal model were evaluated. The data revealed that afCc/Pb (100 µg/ml) exhibited strong anti-atopic activity, causing a significant 40% reduction in immune response, as shown by the extent of ear swelling, resulting from a decrease in the number of eosinophils in the skin tissues due to decreased matrix metalloproteinase-2 and interleukin-31 expression. These results collectively suggest that afCc/Pb has the potential to alleviate the symptoms of atopic dermatitis in a mouse model of DNFB-induced atopic dermatitis, and that it may be a valuable bioresource for the cosmetic/cosmeceutical industry.

Comparison of the toxicity of aqueous and ethanol fractions of Angelica keiskei leaf using the eye irritancy test.

To determine whether aqueous and ethanol fractions of the Angelica keiskei leaf exert toxicity when used for cosmetic purposes, we performed the acute eye irritancy test. Animals were treated with sample fractions (100 mg/dose) according to standard procedure guidelines. No significant changes or damage was detected in the fraction-treated groups in terms of ocular lesions in the cornea, the size of the cornea with turbidity, swelling of the eyelid and emission discharge. However, sodium dioctyl sulfosuccinate, a positive control, induced severe toxic symptoms. Thus, aqueous and ethanol fractions of Angelica keiskei do not appear to induce acute toxicity in the eye lens, as assessed from anatomical and pathological observations in the rabbit eye. Our results collectively suggest that aqueous and ethanol fractions show promise as cosmetic ingredients that do not cause eye toxicity.

Inhibition of xenograft tumor growth in mice by gold nanoparticle-assisted delivery of short hairpin RNAs against Mcl-1L.

A prerequisite for the therapeutic use of small RNAs is the development of a method that can deliver them into animals. Previous studies have shown the capability of functionalized gold nanoparticles to serve as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNAs (shRNAs) into cultured human cells. Here, we report the ability of the gold nanoparticle-assisted gene delivery system (AuNP-GDS) to deliver shRNA to a xenograft tumor in a mouse model. AuNP-GDS delivery of in vitro synthesized shRNA targeted to the Mcl-1L gene knocked down levels of Mcl-1L mRNA and protein by ~36% and ~26%, respectively, which were sufficient to induce apoptosis of the xenograft tumor cells and consequently inhibited the development of the tumor. We demonstrated that our lego-like AuNP-GDS, which can easily load and deliver shRNAs targeted to any gene of interest into living systems, can deliver shRNAs into xenograft tumors, leading to antitumor activity in an animal model.

Functional investigation of residue G791 of Escherichia coli 16S rRNA: implication of initiation factor 1 in the restoration of P-site function.

Using a specialized ribosome system, previous studies have identified G791 in Escherichia coli 16S rRNA as an invariant and essential residue for ribosome function. To investigate the functional role of G791, we searched for multicopy suppressors that partially restored the protein synthesis ability of mutant ribosomes bearing a G to U substitution at position 791 (U791 ribosomes). Analyses of isolated multicopy suppressors showed that overexpression of initiation factor 1 (IF1) enhanced the protein synthesis ability of U791 ribosomes. In contrast, overexpression of initiation factor 2 (IF2) or IF3 did not enhance the protein synthesis ability of wild-type or U791 ribosomes, and overexpression of IF1 did not affect the function of wild-type or mutant ribosomes bearing nucleotide substitutions in other regions of 16S rRNA. Analyses of sucrose gradient profiles of ribosomes showed that overexpression of IF1 marginally enhanced the subunit association of U791 ribosomes and indicated lower binding affinity of U791 ribosomes to IF1. Our findings suggest the involvement of IF1 in the restoration of the P-site function that was impaired by a nucleotide substitution at residue G791.