Growth Inhibition Research Articles

NEDD4L contributes to ferroptosis and cell growth inhibition in esophageal squamous cell carcinoma by facilitating xCT ubiquitination

AbstractThe oncogene xCT plays an indispensable role in tumor growth by protecting cancer cells from oxidative stress and ferroptosis. Emerging evidence indicated xCT function is tightly controlled by posttranslational modifications, especially ubiquitination. However, it still remains unclear what specific regulatory mechanism of xCT by ubiquitin ligases in human cancers. Here, we reported that NEDD4L, an E3 ubiquitin ligases, inhibited esophageal squamous cell carcinoma (ESCC) tumor growth and facilitated ferroptosis by ubiquitination of xCT. NEDD4L expression was declined in ESCC and was associated with tumor invasion, lymph node metastasis and distant metastasis. Silencing NEDD4L triggered ESCC tumor growth. Meanwhile, knock down of NEDD4L prevented the accumulation of ROS, elevated the level of GSH, reduced the content of MDA in ESCC cells, thereby inhibiting ferroptosis. Mechanistically, NEDD4L directly bound to the ∆CT domain of xCT through its WW and HECT domain. More importantly, NEDD4L promoted xCT degradation by facilitating its polyubiquitination in ESCC cells. Collectively, these findings suggest that NEDD4L is crucial in governing the stability of xCT and mediating ferroptosis in ESCC.

Abstract C026: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

Abstract Epithelial-to-mesenchymal transition (EMT) plays important roles during development, injury repair, cancer invasion and metastasis. The heterogeneity of EMT programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-β upon disseminating to distant sites. Through in vivo selection, we established dormant metastasis models from human and mouse early-stage LUAD cell lines. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers, whereas cells derived from overt metastases express high level of the late-stage progenitor marker Sox9. Using a TGF-β signaling reporter, we showed that in the primitive progenitors, TGF-β induces growth inhibition accompanied by a classic EMT response that subsequently transitions into an atypical mesenchymal state of round morphology as the disseminated cells establish dormancy. Prolonged treatment with TGF-β in vitro recapitulates this transition in these primitive progenitors, but not in the late-stage progenitors. Given this dramatic morphological change, we focused on actin-cytoskeleton regulators and found that TGF-β induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-β as a crucial driver of immune evasive metastatic dormancy. Citation Format: Zhenghan Wang, Yassmin Elbanna, Inês Godet, Lila Peters, George Lampe, Yanyan Chen, Joao Xavier, Morgan Huse, Joan Massagué. TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C026.

4-(Pyrazolyl)benzenesulfonamide Ureas as Carbonic Anhydrases Inhibitors and Hypoxia-Mediated Chemo-Sensitizing Agents in Colorectal Cancer Cells.

Hypoxia in tumors contributes to chemotherapy resistance, worsened by acidosis driven by carbonic anhydrases (hCA IX and XII). Targeting these enzymes can mitigate acidosis, thus enhancing tumor sensitivity to cytotoxic drugs. Herein, novel 4-(pyrazolyl)benzenesulfonamide ureas (SH7a-t) were developed and evaluated for their inhibitory activity against hCA IX and XII. They showed promising results (hCA IX: KI = 15.9-67.6 nM, hCA XII: KI = 16.7-65.7 nM). Particularly, SH7s demonstrated outstanding activity (KIs = 15.9 nM for hCA IX and 55.2 nM for hCA XII) and minimal off-target kinase inhibition over a panel of 258 kinases. In NCI anticancer screening, SH7s exhibited broad-spectrum activity with an effective growth inhibition full panel GI50 (MG-MID) value of 3.5 μM and a subpanel GI50 (MG-MID) range of 2.4-6.3 μM. Furthermore, SH7s enhanced the efficacy of Taxol and 5-fluorouracil in cotreatment regimens under hypoxic conditions in HCT-116 colorectal cancer cells, indicating its potential as a promising anticancer agent.

Abstract C041: MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer

Abstract Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Citation Format: Katherine Murphy. MYC and p53 alterations cooperate through VEGF signaling to repress cytotoxic T cell and immunotherapy responses in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C041.

PLLA Porous Scaffold as a 3D Breast Cancer Model to Investigate Drug Resistance.

Multidrug resistance remains one of the major challenges in breast cancer research, often leading to treatment failure. To better understand this mechanism, sophisticated three-dimensional (3D) tumor models are necessary, as they offer several advantages over traditional bidimensional (2D) cultures. In this study, poly-l-lactic-acid porous scaffolds were produced using a thermally induced phase separation technique and employed as 3D models for breast cancer cell lines: MDA-MB-231, MCF-7, and its multidrug-resistant variant, MCF-7R. The MTS assay was used to compare growth inhibition following doxorubicin treatment in 2D and 3D. Remarkably, the IC50 values increased in 3D cultures compared to 2D: MDA-MB-231 (445 vs. 54.5 ng/mL), MCF-7 (7.45 vs. 0.75 μg/mL), and MCF-7R (165 vs. 39 μg/mL). MCF-7R, which usually shows greater resistance in 2D, demonstrated even higher resistance in 3D. In fact, IC50 was not reached within 3 days as with the other models, but only after 6 days. Cellular morphology also played a crucial role. When treated with concentrations higher than the IC50, MDA-MB-231 cells lost their physiological 3D clustered structure, while MCF-7 and its resistant variant exhibited disrupted layers. All cell lines in 3D showed higher chemoresistance, suggesting a more biomimetic spatial architecture. Our work bridges the gap between monolayer and animal models, highlighting the potential of polymeric 3D scaffolds in breast cancer research.

Toxicity assessment of Cd and Cu on physicochemical parameters of green microalga Scenedesmus quadricauda

AbstractDue to immobilization of heavy metals in the environment, it is necessary to evaluate their toxic effects on living cells. In this study, ecotoxicity of Cd and Cu was studied on inhibition of growth, chlorophyll a (Chl a) and chlorophyll b (Chl b), protein thiol groups and changes in bioconcentration factor (BCF). Cadmium toxicity was confirmed to be higher than Cu on Scenedesmus quadricauda. While in the lower concentrations of Cu the specific growth rate (SGR) was increased, in the presence of Cd only inhibition was confirmed. Level of Chl a was decreased more than level of Chl b. Only at low of Cu concentrations up to 0.07 mg L-1 the specific growth rate and both photosynthetic pigments increased compared to control. We observed a high bioaccumulation of Cd and Cu in the cells through bioconcentration values. We determined a negative correlation between protein thiol groups and the Chl a (r=-0.461, p<0.01) and Chl b (r=-0.416, p<0.01), respectively, in Cu presence that indicates higher consumption of protein thiol groups probably due higher level of oxidative stress in the cells. Although, we did not confirm this significant correlation for cells cultivated in the presence of Cd, we found significant negative correlation between Cd accumulation and Chl a (r=-0.643) or Chl b (r=-0.699), respectively. S. quadricauda could be suitable candidate for bioremediation of contaminated waters (e.g. in algae-bacteria consortium) because has high capacity of Cd and Cu bioaccumulation and still have enough protein thiols to protect against damage of possible higher oxidative stress.

Salmonella virulence factors induce amino acid malabsorption in the ileum to promote ecosystem invasion of the large intestine

The gut microbiota produces high concentrations of antimicrobial short-chain fatty acids (SCFAs) that restrict the growth of invading microorganisms. The enteric pathogen Salmonella enterica serovar (S.) Typhimurium triggers inflammation in the large intestine to ultimately reduce microbiota density and bloom, but it is unclear how the pathogen gains a foothold in the homeostatic gut when SCFA-producing commensals are abundant. Here, we show that S. Typhimurium invasion of the ileal mucosa triggers malabsorption of dietary amino acids to produce downstream changes in nutrient availability in the large intestine. In gnotobiotic mice engrafted with a community of 17 human Clostridia isolates, S. Typhimurium virulence factors triggered marked changes in the cecal metabolome, including an elevated abundance of amino acids. In an ex vivo fecal culture model, we found that two of these amino acids, lysine and ornithine, countered SCFA-mediated growth inhibition by restoring S. Typhimurium pH homeostasis through the inducible amino acid decarboxylases CadA and SpeF, respectively. In a mouse model of gastrointestinal infection, S. Typhimurium CadA activity depleted dietary lysine to promote cecal ecosystem invasion in the presence of an intact microbiota. From these findings, we conclude that virulence factor-induced malabsorption of dietary amino acids in the small intestine changes the nutritional environment of the large intestine to provide S. Typhimurium with resources needed to counter growth inhibition by microbiota-derived SCFAs.

Combining the Powerful Antioxidant and Antimicrobial Activities of Pomegranate Waste Extracts with Whey Protein Coating-Forming Ability for Food Preservation Strategies

Different solvents water, ethanol and ethanol/water (6:4 v/v), were compared in the extraction of pomegranate peels and seeds (PPS) in terms of recovery yields, antioxidant properties, and antimicrobial action against typical spoilage bacterial and fungal species. The best performing extract (ethanol/water (6:4 v/v) was shown to contain mostly ellagic acid and punicalagin as phenolic compounds (5% overall) and hydrolysable tannins (16% as ellagic acid equivalents) and was able to inhibit the growth of the acidophilic Alicyclobacillus acidoterrestris at a concentration as low as 1%. The preservation of the organoleptic profile of A. acidoterrestris-inoculated apple juice with extract at 1% over 20 days was also observed thanks to the complete inhibition of bacterial growth, while the extract at 0.1% warranted a significant (40%) inhibition of the enzymatic browning of apple smoothies over the first 30 min. When incorporated in whey proteins’ isolate (WPI) at 5% w/w, the hydroalcoholic extract conferred well appreciable antioxidant properties to the resulting coating-forming hydrogel, comparable to those expected for the pure extract considering the amount present. The WPI coatings loaded with the hydroalcoholic extract at 5% were able to delay the browning of cut fruit by ca. 33% against a 22% inhibition observed with the sole WPI. In addition, the functionalized coating showed an inhibition of lipid peroxidation of Gouda cheese 2-fold higher with respect to that observed with WPI alone. These results open good perspectives toward sustainable food preservation strategies, highlighting the potential of PPS extract for the implementation of WPI-based active packaging.

Inhibition of NPC1 suppresses cell proliferation and β-catenin signaling activation of liver cancer.

Niemann-Pick Type C1 (NPC1) plays a significant role in the development of liver diseases and liver cancer. Our objective was to investigate the involvement of NPC1 in regulating liver cancer development. We observed that high levels of NPC1 expression in tumor tissues from patients with liver cancer were associated with a poor prognosis. Through in vitro experiments, we found that inhibiting NPC1 expression reduced the proliferation, invasion, and migration of liver cancer cells, while also inducing apoptosis in these cells. Additionally, the inhibition of NPC1 led to decreased activation of Wnt/β-catenin signaling. In vivo studies further supported our findings by demonstrating that the suppression of liver cancer cell growth was effectively achieved through the inhibition of NPC1. Overall, our results strongly indicate that the inhibition of NPC1 suppresses liver cancer cell proliferation. Targeting NPC1 is a promising potential therapeutic strategy for liver cancer.

The extended recovery ring-stage survival assay is a scalable alternative for artemisinin susceptibility phenotyping of fresh Plasmodium falciparum isolates.

Artemisinin partial resistance (ART-R) has emerged in eastern Africa, necessitating regular surveillance of susceptibility of Plasmodium falciparum to artemisinins. The microscopy-based ring-stage survival assay (RSA) provides a laboratory correlate of ART-R but is limited by low throughput and subjectivity of microscopic counts of viable parasites. The extended recovery ring-stage survival assay (eRRSA) replaces microscopy with efficient quantitative PCR (qPCR) readouts but has been studied only with culture-adapted P. falciparum clones. We measured susceptibility to dihydroartemisinin (DHA) after a 6-h incubation with 700-nM DHA, followed by culture without drug, by comparing survival with that of untreated controls by microscopy (the RSA) or qPCR (the eRRSA) and also performed standard growth inhibition (half-maximal inhibitory concentration [IC50]) assays for 122 P. falciparum isolates freshly collected in eastern and northern Uganda from March to July 2022. The median values for RSA survival, eRRSA fold change, and DHA IC50 were 3.0%, 46.2, and 3.2 nM, respectively. RSA percent survival and eRRSA fold changes correlated strongly (Spearman correlation coefficient [rs] = -0.7411, P < 0.0001), with modest associations between the presence of validated P. falciparum Kelch13 ART-R mutations (C469Y or A675V) and RSA (median survival 2.6% for wild type [WT] vs 4.1% for mutant, P = 0.01), or eRRSA (median fold change 63.4 for WT vs 30.9 for mutant, P = 0.003) results. Significant correlations were also observed between DHA IC50 values and both RSA percent survival (rs = 0.4235, P < 0.0001) and eRRSA fold changes (rs = -0.4116, P < 0.0001). The eRRSA is a scalable alternative for phenotyping fresh P. falciparum isolates, providing similar results with improved throughput.

INFLUENCE OF ALTERNARIA SPP. FUNGI METABOLITES ON DEVELOPMENT OF WHEAT SPROUTS

The study purpose was in vitro investigation of phytotoxic activity of culture broth filtrates (CBFs) of five Alternaria Spp. fungi isolates on development of wheat sprouts. Fungi isolates were received from wheat seeds cultivated under conditions of Northern Kazakhstan. According to the study findings, culture filtrates of all the isolates studied varied with regard to their influence on wheat sprout development. Stimulant effects on seed germinating power were revealed with 14- and 21-day CBFs of A. infectoria (7/4), 14-day CBF of A. brassicae (7/2) and 21-day CBF of A. porri (8/1). It was found that long-term cultivation of fungi on a culture medium contributed to media pH modification towards alkalization due to production of metabolites of various chemical nature, including toxins, which could trigger growth inhibition of radicles and sprouts. However, the CBFs were predominantly low-toxic or non-toxic. Under effect of the 14-day CBF, significant positive correlation was identified between pH values and sprout lengths (0,86±0,25), while negative correlation was found between pH values and germinating power (-0,75±0,33). Change of pH value on the 21st day significantly affected radicle length (-0,92±0,20). Based on the findings it can be concluded that the 14-day CBF of all the Alternaria fungi isolates studied was most toxic for wheat sprouts.

Collembola growth in heavy metal-contaminated soils.

Collembola play a key role in soil ecosystems by decomposing organic matter. Most of them inhabit the upper layers of the soil and are susceptible to contamination present in the pore water. These arthropods serve as model organisms in ecotoxicology for short and long-term exposure. This study aimed to assess the toxicity of three heavy metals (lead [Pb], cadmium [Cd], and copper [Cu]) using the springtail species Folsomia candida as the test organism, with mortality and growth inhibition as the measure of toxicity. We hypothesised that increasing metal concentrations in the soil would correspond to a growth reduction of Collembola. Each heavy metal was tested at a minimum of eight increasing concentrations in six replications. Twenty 10-12-day-old individuals were introduced into each test container filled with contaminated or control soil and incubated for 14 days. The test endpoints included growth inhibition determined by comparing F. candida growth rates in contaminated soil with those in control soil, as well as mortality rates. The EC50 values (mg/kg) for heavy metals were as follows: Cd = 66.89, Cu = 791.01, Pb = 10075.48. Our findings suggest that growth inhibition is a reliable indicator of Collembola toxicity to heavy metals.

Abstract B026: Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer

Abstract Introduction: A major contributor to prostate cancer (PCa) mortality is the development of resistance to single agents targeting androgen receptor (AR) signaling. We have developed an unbiased shRNA-based negative selection screen to identify small RNAs (shRNAs and siRNAs) that inhibit PCa cell growth and survival (toxic sRNAs). Computational analyses of the most toxic sRNAs identified revealed that each can simultaneously target multiple known AR-coregulatory mRNAs. AR-coregulators modulate AR transcriptional response and are essential to AR signaling. We have proposed that the identified toxic sRNAs inhibit androgen signaling through seed-mediated targeting of androgen regulatory gene networks, resulting in inhibition of cell growth and viability. The goal of the current studies was to begin to analyze the broad therapeutic potential of these toxic sRNAs for PCa. We focused on analyzing i) their potential to thwart cell growth and/or viability when delivered to preclinical models of PCa, and ii) the targetome of the toxic sRNAs as a mean to uncover novel AR-coregulators that represent a targetable axis within androgen signaling. Methods: i) cationic lipid nanoparticles (LNPs), that efficiently deliver the toxic siRNAs to PCa cell lines, were used to deliver toxic siRNAs to organoids grown from PCa patient derived xenografts (PDX) models. 3D image analysis using the Ominer® software package was used to determine organoid count and size, the numbers of nuclei per organoid, and the fraction of dead cells to discriminate between cytotoxic and cytostatic effects. ii) to identify novel AR-coregulators, toxic siRNA direct targets were identified by sequencing of transcripts captured by biotinylated toxic siRNAs. Directly bound targets, were subjected to pathway enrichment and network analysis followed by identification of hub genes (Cytoscape). The effect of individually silencing potential AR-coregulators on the expression of androgen responsive genes was analyzed using Nanostring. Results and Conclusions: i) The effect of the toxic siRNAs on growth and viability of the PCa PDX-derived organoids will be presented and discussed as compelling preliminary data for therapeutic testing in more complex pre-clinical models (PCa PDXs). ii) Based on bioinformatic and downstream analysis of the direct targets of two toxic siRNAs, we selected 12 potentially novel AR- coregulators, with varied known functions including chromatin modifiers, transcriptional regulators, etc. Silencing of several of these potential AR-coregulators significantly affect expression of numerous AR responsive genes. Future experiments will focus on analyzing their interaction and functional association with the AR (i.e. effect of AR binding to chromatin). In conclusion, we have developed an assay that allowed the identification of toxic sRNAs for PCa cells and demonstrated their potential as therapeutics and as a tool for discovery of potential novel targets for PCa. Citation Format: Joshua M Corbin, Hamilton Young, Stefan Wilhelm, Adam S Asch, Chao Xu, Maria J Ruiz Echevarria. Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B026.

DDX3 participates in miRNA biogenesis and RNA interference through translational control of PACT and interaction with AGO2.

DDX3 is a DEAD-box RNA helicase that plays multiple roles in RNA metabolism, including translation. We previously reported that DDX3 is required for translation of PACT, a binding partner of Dicer, suggesting a role for DDX3 in microRNA (miRNA) biogenesis and RNA interference (RNAi). Emerging evidence suggests that DDX3 plays a vital role in tumorigenesis and cancer progression, however, its underlying mechanism is still not fully understood. Here, we showed that the control of PACT by DDX3 is conserved in human cells and Caenorhabditis elegans. Using a miRNA microarray, we found that DDX3 regulates the expression of a small subset of cancer-related miRNAs. These oncogenic miRNAs were down-regulated by knockdown of DDX3 or PACT and up-regulated by overexpression of DDX3 or PACT in HEK293Tcells. Similar results were obtained in human cancer HCT116 and HeLa cells. Dual luciferase reporter assay showed that DDX3 and PACT are required for short hairpin RNA (shRNA)-induced RNAi. We also performed co-immunoprecipitation to confirm the interaction between DDX3 and AGO2, a significant component of the RNA-induced silencing complex, supporting a role for DDX3 in the RNAi pathway. We further examined the effects of DDX3 and PACT on cell proliferation, and stable overexpression of DDX3 in HEK293 cells results in loss of contact inhibition of cell growth. Hence, we propose that DDX3 may participate in cancer development by regulating the RNAi pathway.

Impact of spot blotch pathogen Bipolaris sorokiniana on important seed quality parameters in wheat and its management with biocontrol agents and newer fungicide molecules.

Spot blotch disease is prominent amongst several biotic stresses challenging wheat yield and quality. The impact of seed inoculation with Bipolaris sorokiniana, on important wheat seed quality of popular cultivar (cv.) DBW 187, was estimated in comparison to susceptible check WH 147. DBW 187 responded very effectively against pathogen keeping all parameters well ahead of susceptible check. The variety could resist declination in per cent germination with 8.07% in comparison to WH 147 which resulted in 14.72% reduction in germination after pathogen inoculation. Similarly, after pathogen's inoculation, speed of germination (80.06) and vigour index I (2677) and II (24379) were significantly higher in cv. DBW 187 as against cv. WH 147, which resulted in lower speed of germination and vigour index I and II, i.e. 74.37, 2249, and 16715, respectively. Amongst the biocontrol agents, Trichoderma harzianum was found to be the most effective in managing this pathogen, which caused 86.30% inhibition in mycelial growth of B. sorokiniana. Amongst the seven fungicides used to manage Bipolaris sorokiniana in vitro, tebuconazole 50% + trifloxystrobin 25% WG, propiconazole 25% EC both at 75 and 100ppm, and kresoxim methyl 44.3% SC at 100ppm were most effective against Bipolaris sorokiniana, causing complete inhibition in its radial growth. Conclusively, wheat cv. DBW 187 has capacity to resist seed health deterioration caused by the pathogen. Out of seven evaluated fungicides, the best three newer fungicide molecules, viz., tebuconazole 50% + trifloxystrobin 25% WG, propiconazole 25% EC, and kresoxim methyl 44.3% SC, can judiciously be utilized in combination with biocontrol agents, Trichoderma harzianum, T. asperellum, and Chaetomium globosum, keeping the fungicide load minimum for the effective management of B. sorokiniana.

Abstract I008: Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology

Abstract Many potential oncology targets are difficult to modulate by conventional classes of agents. Interfering with these targets at the level of RNA is a novel approach for difficult-to target proteins, as well as for non-coding RNAs, emerging targets that are part of the “dark matter’ of the genome. Antisense oligonucleotide (ASO) therapeutics are short sequences of 12- 30 DNA/RNA residues, designed to bind the cognate transcribed RNA. The ASO is chemically modified to increase stability, improve binding affinity, optimize pharmacokinetic properties and reduce immunostimulatory potential. The hybridization of ASO to cognate RNA results in cleavage of bound RNA by RNAse H1 and subsequent degradation. Several ASOs are marketed for treatment of rare diseases, but to date none have been approved for oncology indications. The long-noncoding RNA (lncRNA) MALAT1 is an example of an oncology target uniquely suited for an ASO therapeutic. This lncRNA is not translated and resides mostly in the nucleus, limiting access for most therapeutic approaches. MALAT1 is highly upregulated in various cancers, appears to play a role in epithelial to mesenchymal transition, and is associated with disease progression in patients. Inhibition of MALAT1 in preclinical breast cancer models, either genetic or pharmacologic using a murine ASO, leads to changes in tumor architecture and reduction in metastasis. FLM-7523 is a 16-mer ASO designed to bind to human MALAT1. It is a generation 2.5 design based on the constrained ethyl (cEt) modification to the 3 residues on the 3’ and 5’ ends of the ASO, and administered in a saline solution without any delivery vehicle. Preclinical studies have shown that FLM-7523 effectively inhibits MALAT1 expression in in vitro and in vivo models, and GLP toxicology studies have been completed to support a First-in-Human clinical trial. The signal transduction molecule STAT3 is another oncology target of interest that has, to date, been difficult to modulate. Danvatirsen is a 16-mer generation 2.5 ASO targeting human STAT3 mRNA and is currently in clinical trials. Preclinical in vivo studies in murine tumor models have demonstrated a reduction in STAT3 mRNA and protein following treatment with a STAT3 targeting ASO, along with tumor growth inhibition. Combination treatments with immune checkpoint inhibitors, including anti-PD-1 and anti-PD-L1, demonstrate improved tumor growth inhibition relative to monotherapy. In clinical trials, tumor uptake of danvatirsen is observed in post-treatment biopsies, with corresponding reduction in STAT3 protein. Danvatirsen is generally well tolerated with the most common adverse events being increased liver function tests and decreased myeloid hematologic parameters, both of which are easily monitored, manageable, and reversible. Clinical responses have been observed with the most robust being in combination with a PD-L1 inhibitor in recurrent/metastatic head and neck squamous cell carcinoma. In conclusion, ASOs are novel therapies that can inhibit oncology targets not tractable by traditional approaches. Citation Format: Andrew Denker. Antisense Oligonucleotides as Therapeutics for Difficult-to-Drug Targets in Oncology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I008.

The preliminary study of antifungal and antibacterial properties of wine by-products

Background: Nowadays, the search for novel alternative antimicrobials has gained significant popularity. This surge is driven by the vast array of adverse effects of synthetic antimicrobials on human health and the environment. Consequently, the study of new natural sources of comparably ecologically safe and simultaneously effective antimicrobials is extremely important. In this context, the interest in wine by-products is growing due to their diverse beneficial properties. Traditionally considered industrial waste, wine by-products now offer promising opportunities. Notably, the unique climatic and geographical features of Armenian black grape varieties, such as Areni sev, make their by-products an attractive subject for study, with potential applications in agriculture. Objective: The study aims to investigate the antifungal and antibacterial properties of wine by-products obtained from the Areni sev grape variety. Methods: For the extraction of bioactive compounds, frozen and dry samples were homogenized. The maceration was performed in 70% hydroethanolic solution after incubation on a magnetic stirrer at room temperature. The obtained extracts were filtered through a 0.45 µm filter and evaluated for antifungal and antibacterial activity by disco-diffusion method. Results: Hydroethanolic extracts of wine by-products have exhibited antifungal and antibacterial activities. Specifically, the frozen samples demonstrated low antifungal activity against the Penicillium canescens with a 7.5 mm growth inhibition zone. In contrast, dried samples showed no antifungal activity. The highest antifungal activity was observed against Aspergillus niger with growth inhibition zones of 18 mm (frozen samples) and 15 mm (dried samples), respectively. Regarding antibacterial activity, wine by-product hydroethanolic extracts showed extremely low efficacy. Notably, frozen samples exhibited an 8 mm growth inhibition zone against Curtobacterium flaccumfaciens and 6.35 mm against Xanthomonas vesicatoria. Extracts from dried samples demonstrated a 6.35 mm inhibition zone against Curtobacterium flaccumfaciens and a 7 mm growth inhibition zone against Xanthomonas vesicatoria. Conclusion: Summarizing the results of the conducted experiments, it can be concluded that the used wine by-product derived from Areni sev grape has demonstrated notable antimicrobial activities, with a stronger emphasis on antifungal properties. The extracts obtained from frozen samples exhibited greater efficacy than those derived from dried samples. Future research plans include a more detailed investigation of Areni sev grape wine by-products. Keywords: wine by-product, grape pomace, Areni sev, bioactive compounds, antimicrobial properties

Enhancing the anticancer effect of metformin through nanoencapsulation: Apoptotic induction, inflammatory reduction, and suppression of cell migration in colorectal cancer cells.

Colorectal cancer (CRC) continues to be a significant health challenge, necessitating the development of efficient therapeutic strategies. Drug repurposing, which involves the use of existing medications for new purposes, presents a promising opportunity. Metformin, a widely used antidiabetic drug, has demonstrated potential anticancer effects. To enhance its efficacy, we formulated nano-metformin, metformin encapsulated within pectin nanoparticles. Our study aimed to evaluate the superiority of nano-metformin over free metformin in treating CRC. The cytotoxicity of both metformin and nano-metformin on Caco-2 CRC cells was assessed using the MTT assay, revealing a significant dose-dependent inhibition of cell growth using nano-metformin. The anti-inflammatory potential was evaluated by measuring the levels of nitric oxide and the pro-inflammatory cytokines IL-2 and IL-6 following lipopolysaccharide (LPS) induction, and the results revealed that treating LPS-induced cells with nano-metformin significantly reduced the production of these inflammatory mediators. To elucidate the mechanism of cell death, we employed an acridine orange/ethidium bromide staining assay, which revealed the enhancement of apoptotic cell death following treatment with nano-metformin. Additionally, we examined the expression of key apoptotic regulators using real-time qPCR. Nano-metformin, in particular, significantly downregulated the expression of the antiapoptotic markers Bcl-2 and Survivin while upregulating the proapoptotic caspases 3, 7, and 9. The comet assay revealed significant DNA damage induced by treatment with the nano-metformin compared with that in the free form. Moreover, nano-metformin significantly reduced the migration ability of cells. In conclusion, our work revealed the superior efficacy of our formulated nanoform over free metformin, highlighting its potential as a promising therapeutic agent for CRC treatment.

Abstract B026: Glioblastoma multiforme disseminates outside the central nervous system

Abstract Introduction: The liquid biopsy (LB) concept has gained significant attention in the last decade and has the potential to revolutionize cancer care. However, more validation in clinical trials is required on the value of LB in medical settings. Here, we focus on glioblastoma multiforme (GBM), a highly invasive and fatal subtype of the central nervous system malignant tumors. Circulating tumor cells (CTCs) are primary tumor cells released into the peripheral blood and migrating to distant organs. CTC presence is related to tumor spread and recurrence. In GBM, factors like short patient survival, the blood-brain barrier (BBB), and lack of lymphatic drainage in brain tissue make systemic dissemination rare but possible. Thus, noninvasive CTC detection can hold potential for diagnostic, predictive, and prognostic applications. Material and Methods: For cell stability preservation before sample processing, the peripheral blood samples from curatively resected GBM patients were collected preoperatively in Cell-Free DNA BCT® tubes (Streck, Inc.). CTCs were identified using CytoTrack CT11TM (2/C), a semi-automated immunofluorescence microscopy using glial fibrillary acidic protein, vimentin, and CD45 immunostaining, and DAPI counterstaining. The preliminary Kaplan-Maier survival analysis based on CTC positivity was performed in 39 patients with a follow-up longer than two years. Results: We analyzed the CTC presence in 100 GBM patient peripheral blood samples. The CTCs or cancer cell clusters were found in 35% of GBM patients. The Kaplan-Maier survival analysis did not show a correlation between CTC presence and survival. This phenomenon may be associated with the generally short survival of these patients, which is expected to increase with advances in oncology and palliative care and/or a possible suppression of tumor cell growth outside the CNS. Conclusions: We have observed the CTCs in the peripheral blood of GBM patients. The patient enrollment and clinical data recording are still ongoing. Citation Format: Pavel Stejskal, Josef Srovnal, Alona Rehulkova, Ondrej Kalita, Marek Slachta, Marian Hajduch. Glioblastoma multiforme disseminates outside the central nervous system [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B026.

Cationic Metal-Organic Layer Delivers siRNAs to Overcome Radioresistance and Potentiate Cancer Radiotherapy.

Radiotherapy plays an important role in modern oncology, but its treatment efficacy is limited by the radioresistance of tumor cells. As a member of the inhibitor of apoptosis protein family, survivin plays a key role in developing radioresistance by mediating apoptosis evasion, promoting epithelial-mesenchymal transition, and modulating cell cycle dynamics. Efficient downregulation of survivin expression presents a promising strategy to enhance the antitumor effects of radiotherapy. Herein, we report the design of a hafnium-porphyrin-based cationic metal-organic layer (CMOL) with quaternary ammonium capping groups to deliver small interfering RNAs (siRNAs) for enhanced radiotherapy. The CMOL@siRNA nanoplatform not only increased energy deposition from X-rays and reactive oxygen species generation via a unique radiotherapy-radiodynamic therapy process, but also effectively delivered siRNAs to downregulate survivin expression and ameliorate radioresistance of cancer cells. Consequently, CMOL@siRNA in combination with low-dose X-ray irradiation demonstrated remarkable antitumor efficacy with 96.9% and 91.4% tumor growth inhibition in murine colorectal carcinoma and triple-negative breast cancer models, respectively.