Reduced Tumor Incidence Research Articles

A Biomimetic Autophagosomes-Based Nanovaccine Boosts Anticancer Immunity.

Personalized cancer vaccines based on tumor cell lysates offer promise for cancer immunotherapy yet fail to elicit a robust therapeutic effect due to the weak immunogenicity of tumor antigens. Autophagosomes, obtained from pleural effusions and ascites of cancer patients, have been identified as abundant reservoirs of tumor neoantigens that exhibit heightened immunogenicity. However, their potential as personalized cancer vaccines have been constrained by suboptimal lymphatic-targeting performances and challenges in antigen-presenting cell endocytosis. Here,a reinforced biomimetic autophagosome-based (BAPs) nanovaccine generated by precisely amalgamating autophagosome-derived neoantigens and two types of adjuvants capable of targeting lymph nodes is developed to potently elicit antitumor immunity. The redox-responsive BAPs facilitate cytosolic vaccine opening within antigen-presenting cells, thereby exposing adjuvants and antigens to stimulate a strong immune response. BAPs evoke broad-spectrum T-cell responses, culminating in the effective eradication of 71.4% of established tumors. Notably, BAPs vaccination triggers enduring T-cell responses that confer robust protection, with 100% of mice shielded against tumor rechallenge and a significant reduction in tumor incidence by 87.5%. Furthermore, BAPs synergize with checkpoint blockade therapy to inhibit tumor growth in the poorly immunogenic breast cancer model. The biomimetic approach presents a powerful nanovaccine formula with high versatilityfor personalized cancer immunotherapy.

A probiotic Limosilactobacillus fermentum GR-3 mitigates colitis-associated tumorigenesis in mice via modulating gut microbiome

Bacterial therapy for colorectal cancer (CRC) represents a burgeoning frontier. The probiotic Limosilactobacillus fermentum GR-3, derived from traditional food “Jiangshui”, exhibited superior antioxidant capacity by producing indole derivatives ICA and IPA. In an AOM/DSS-induced CRC mouse model, GR-3 treatment alleviated weight loss, colon shortening, rectal bleeding and intestinal barrier disruption by reducing oxidative stress and inflammation. GR-3 colonization in distant colon induced apoptosis and reduced tumor incidence by 51.2%, outperforming the control strain and vitamin C. The beneficial effect of GR-3 on CRC was associated with gut microbiome modulation, increasing SCFA producer Lachnospiraceae NK4A136 group and suppressing pro-inflammatory strain Bacteroides. Metagenomic and metabolic analyses revealed that GR-3 intervention upregulated antioxidant genes (xseA, ALDH) and butyrate synthesis gene (bcd), while increasing beneficial metabolites (SCFAs, ICA, IPA, VB12 and VD3) and reducing harmful secondary bile acids. Overall, GR-3 emerges as a promising candidate in CRC therapy, offering effective gut microbiome remediation.

Development and evaluation of imiquimod-loaded nanoemulsion-based gel for the treatment of skin cancer

BackgroundThe human skin, as the body’s largest organ, is particularly sensitive to many chemical mutagens and carcinogens encountered in daily life. Skin cancer has become a notable global health concern, partly due to increased exposure to environmental pollutants and UV rays. Various treatments are available to treat skin cancer. Imiquimod is approved for the treatment of actinic keratosis and basal cell carcinoma. The present investigation aimed to develop nanoemulsion-based gel with imiquimod (2.5% w/w) and carbopol ultrez 10 NF using a modified method to enhance the solubility, permeation, and therapeutic effectiveness of imiquimod to treat skin cancer. Combinations of rose oil and oleic acid, with Tween 20/Propylene glycol as Smix, were used in the formulation. The formulation underwent evaluation for parameters such as % drug content, in vitro drug diffusion studies, viscosity, skin irritation, in vitro cytotoxicity assay (MTT assay) and the DMBA/ croton oil skin cancer in vivo model.ResultsThe formulation showed a minimum globule size of 118 nm, a zeta potential– 56.26 mV, a PDI of 0.378 and a drug content of 99.77%. In vitro drug release exhibited 45.00% of imiquimod release within 8 h, while approximately 34.32% release was found from the commercial cream. The imiquimod-loaded nanoemulsion-based gel showed significant cytotoxicity (p < 0.001) against the A431 cell line compared to Imiquad cream. The IC50 value of the imiquimod-loaded nanoemulsion-based gel was noted to be 10.76 ± 2.54 µg/mL. In vivo results showed a significant reduction in tumor incidence (16.66%), tumor volume (140.26 ± 3.48 mm3), tumor burden (5.50 mm3) and tumor mass (0.66 ± 0.05 g) compared with the DMBA/croton oil carcinogen treatment control group. Histopathological finding showed the absence of keratinized pearls, epidermal hyperplasia, and acanthosis in the formulation treated group.ConclusionThe results revealed that the nanoemulsion-based gel, with half the IMQ concentration of the commercial cream and incorporating Carbopol Ultrez 10NF, is a promising method for treating skin carcinogenesis. It potentially reduces dose-dependent side effects and demonstrating enhanced efficacy.Graphical abstract

A novel combinatorial approach usingsulforaphane- and withaferin A-rich extracts for prevention of estrogen receptor-negative breast cancer through epigenetic and gut microbial mechanisms.

Estrogen receptor-negative [ER(-)] mammary cancer is the most aggressive type of breast cancer (BC) with higher rate of metastasis and recurrence. In recent years, dietary prevention of BC with epigenetically active phytochemicals has received increased attention due to its feasibility, effectiveness, and ease of implementation. In this regard, combinatorial phytochemical intervention enables more efficacious BC inhibition by simultaneously targeting multiple tumorigenic pathways. We, therefore, focused on investigation of the effect of sulforaphane (SFN)-rich broccoli sprouts (BSp) and withaferin A (WA)-rich Ashwagandha (Ash) combination on BC prevention in estrogen receptor-negative [ER(-)] mammary cancer using transgenic mice. Our results indicated that combinatorial BSp + Ash treatment significantly reduced tumor incidence and tumor growth (~ 75%) as well as delayed (~ 21%) tumor latency when compared to the control treatment and combinatorial BSp + Ash treatment was statistically more effective in suppressing BC compared to single BSp or Ash intervention. At the molecular level, the BSp and Ash combination upregulated tumor suppressors (p53, p57) along with apoptosis associated proteins (BAX, PUMA) and BAX:BCL-2 ratio. Furthermore, our result indicated an expressional decline of epigenetic machinery HDAC1 and DNMT3A in mammary tumor tissue because of combinatorial treatment. Interestingly, we have reported multiple synergistic interactions between BSp and Ash that have impacted both tumor phenotype and molecular expression due to combinatorial BSp and Ash treatment. Our RNA-seq analysis results also demonstrated a transcriptome-wide expressional reshuffling of genes associated with multiple cell-signaling pathways, transcription factor activity and epigenetic regulations due to combined BSp and Ash administration. In addition, we discovered an alteration of gut microbial composition change because of combinatorial treatment. Overall, combinatorial BSp and Ash supplementation can prevent ER(-) BC through enhanced tumor suppression, apoptosis induction and transcriptome-wide reshuffling of gene expression possibly influencing multiple cell signaling pathways, epigenetic regulation and reshaping gut microbiota.

Exosome-like Systems: From Therapies to Vaccination for Cancer Treatment and Prevention-Exploring the State of the Art.

Cancer remains one of the main causes of death in the world due to its increasing incidence and treatment difficulties. Although significant progress has been made in this field, innovative approaches are needed to reduce tumor incidence, progression, and spread. In particular, the development of cancer vaccines is currently ongoing as both a preventive and therapeutic strategy. This concept is not new, but few vaccines have been approved in oncology. Antigen-based vaccination emerges as a promising strategy, leveraging specific tumor antigens to activate the immune system response. However, challenges persist in finding suitable delivery systems and antigen preparation methods. Exosomes (EXs) are highly heterogeneous bilayer vesicles that carry several molecule types in the extracellular space. The peculiarity is that they may be released from different cells and may be able to induce direct or indirect stimulation of the immune system. In particular, EX-based vaccines may cause an anti-tumor immune attack or produce memory cells recognizing cancer antigens and inhibiting disease development. This review delves into EX composition, biogenesis, and immune-modulating properties, exploring their role as a tool for prevention and therapy in solid tumors. Finally, we describe future research directions to optimize vaccine efficacy and realize the full potential of EX-based cancer immunotherapy.

Oral intake of bucillamine, carvedilol, metformin, or phenformin does not protect against UVR-induced squamous cell carcinomas in hairless mice

Squamous cell carcinoma represents the second most common type of keratinocyte carcinoma with ultraviolet radiation (UVR) making up the primary risk factor. Oral photoprotection aims to reduce incidence rates through oral intake of photoprotective compounds. Recently, drug repurposing has gained traction as an interesting source of chemoprevention. Because of their reported photoprotective properties, we investigated the potential of bucillamine, carvedilol, metformin, and phenformin as photoprotective compounds following oral intake in UVR-exposed hairless mice. Tumour development was observed in all groups in response to UVR, with only the positive control (Nicotinamide) demonstrating a reduction in tumour incidence (23.8%). No change in tumour development was observed in the four repurposed drug groups compared to the UV control group, whereas nicotinamide significantly reduced carcinogenesis (P = 0.00012). Metformin treatment significantly reduced UVR-induced erythema (P = 0.012), bucillamine and phenformin increased dorsal pigmentation (P = 0.0013, and P = 0.0005), but no other photoprotective effect was observed across the repurposed groups. This study demonstrates that oral supplementation with bucillamine, carvedilol, metformin, or phenformin does not affect UVR-induced carcinogenesis in hairless mice.Graphical

A novel intranasal peptide vaccine inhibits non-small cell lung cancer with KRAS mutation.

KRAS mutations occur commonly in the lung and can lead to the development of non-small cell lung cancer (NSCLC). While the mutated KRAS protein is a neoantigen, it usually does not generate an effective anti-tumor immune response on mucosal/epithelial surfaces. Despite this, mutated KRAS remains a potential target for immunotherapy since immune targeting of this protein in animal models has been effective at eliminating tumor cells. We attempted to develop a KRAS vaccine using mutated and wild-type KRAS peptides in combination with a nanoemulsion (NE) adjuvant. The efficacy of this approach was tested in an inducible mutant KRAS-mouse lung tumor model. Animals were immunized intranasally using NE with KRAS peptides. These animals had decreased CD4+FoxP3+ T cells in both lymph nodes and spleen. Immunized animals also showed higher IFN-γ and IL-17a levels to mutated KRAS that were produced by CD8+ T cells and enhancement in KRAS-specific Th1 and Th17 responses that persisted for 3 months after the last vaccination. Importantly, the immunized animals had significantly decreased tumor incidence compared to control animals. In conclusion, a mucosal approach to KRAS vaccination demonstrated the ability to induce local KRAS-specific immune responses in the lung and resulted in reduced tumor incidence.

Life-long consumption of high level of fruits and vegetables reduces tumor incidence and extends median lifespan in mice.

Epidemiological studies suggest that consumption of fruits and vegetables (FV) is negatively associated with the incidence of certain cancers and mortality. However, a causal relationship has not been demonstrated. Thus, we investigated the effect of life-long consumption of high level of FV on median lifespan, key biological functions, and pathologies in mice fed low-fat (LF) or high-fat (HF) diets and the underlying mechanisms. Using a 2 × 2 factorial design, 5 weeks-old male C57BL/6J mice were randomly assigned to one of four groups (n = 60/group): LF (LF-C, 10% kcal fat), HF (HF-C, 45% kcal fat) or each supplemented with 15% (w/w) of a unique FV mixture (LF + FV and HF + FV, respectively). Mice were euthanized when one group reached 50% mortality. Body weight and composition, tumor incidence, and death were monitored. Blood levels of lipids and pro-inflammatory cytokines were assessed. After 21 months of feeding, HF-C group reached 50% mortality, at which time mice in all groups were terminated. HF-C had higher mortality (50.0%) compared to the LF-C group (18.3%, p = 0.0008). Notably, HF-FV had lower mortality (23.3%) compared to HF-C group (p = 0.008); there was no significant difference in mortality between HF-FV and LF-C groups. Tumors were found in all groups, and were predominantly present in the liver, followed by those of lung, intestine, and seminal vesicle. Tumor incidence in the HF-C group (73.3%) was higher than that in LF-C group (30.0%, p < 0.0001). HF + FV group had 23.3% lower tumor incidence compared to the HF-C group (p = 0.014). No significant difference in tumor incidence between the LF-C and LF + FV groups was observed. Long-term FV supplementation reduced systemic inflammation and blood lipids. We provide the first causal evidence that life-long intake of a diet, containing a high level and large variety of FV, decreases tumor incidence and extends median lifespan in mice fed a western-style high-fat diet. These effects of FV are at least in part due to reduced blood levels of pro-inflammatory cytokines and improved dyslipidemia.

Striking efficacy of a vaccine targeting TOP2A for triple-negative breast cancer immunoprevention

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has a poor prognosis. TOP2A is a key enzyme in DNA replication and is a therapeutic target for breast and other cancers. TOP2A-specific Th1-promoting epitopes with optimal binding affinity to MHC II were identified using a combined scoring system. The multi-peptide TOP2A vaccine elicited a robust immunologic response in immunized mice, as demonstrated by the significant production of Th1 cytokines from immunized animals’ splenocytes stimulated in vitro with TOP2A peptides. Anti-tumor efficacy of the TOP2A vaccine was demonstrated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to adjuvant control. In a genetically engineered mouse (GEM) model of TNBC, vaccinated animals demonstrated a significant reduction in tumor incidence and average tumor volume compared to adjuvant control. Finally, we examined TCR sequences in CD4 tumor Infiltrating lymphocytes (TIL) from vaccinated mice and found that the TIL contained TCR sequences specific to the three vaccine peptides. These data indicate that our newly developed multi-peptide TOP2A vaccine is highly immunogenic, elicits TILs with vaccine specific TCRs, and is highly effective in preventing and intercepting TNBC development and progression in vivo.

Effect of Pre-Treatment with a Recombinant Chicken Interleukin-17A on Vaccine Induced Immunity against a Very Virulent Marek's Disease Virus.

The host response to pathogenic microbes can lead to expression of interleukin (IL)-17, which has antimicrobial and anti-viral activity. However, relatively little is known about the basic biological role of chicken IL-17A against avian viruses, particularly against Marek's disease virus (MDV). We demonstrate that, following MDV infection, upregulation of IL-17A mRNA and an increase in the frequency of IL-17A+ T cells in the spleen occur compared to control chickens. To elaborate on the role of chIL-17A in MD, the full-length chIL-17A coding sequence was cloned into a pCDNA3.1-V5/HIS TOPO plasmid. The effect of treatment with pcDNA:chIL-17A plasmid in combination with a vaccine (HVT) and very virulent(vv)MDV challenge or vvMDV infection was assessed. In combination with HVT vaccination, chickens that were inoculated with the pcDNA:chIL-17A plasmid had reduced tumor incidence compared to chickens that received the empty vector control or that were vaccinated only (66.6% in the HVT + empty vector group and 73.33% in HVT group versus 53.3% in the HVT + pcDNA:chIL-17A). Further analysis demonstrated that the chickens that received the HVT vaccine and/or plasmid expressing IL-17A had lower MDV-Meq transcripts in the spleen. In conclusion, chIL-17A can influence the immunity conferred by HVT vaccination against MDV infection in chickens.

TYMS promotes genomic instability and tumor progression in Ink4a/Arf null background

We previously showed that elevated TYMS exhibits oncogenic properties and promotes tumorigenesis after a long latency, suggesting cooperation with sequential somatic mutations. Here we report the cooperation of ectopic expression of human TYMS with loss of Ink4a/Arf, one of the most commonly mutated somatic events in human cancer. Using an hTS/Ink4a/Arf−/− genetically engineered mouse model we showed that deregulated TYMS expression in Ink4a/Arf null background accelerates tumorigenesis and metastasis. In addition, tumors from TYMS-expressing mice were associated with a phenotype of genomic instability including enhanced double strand DNA damage, aneuploidy and loss of G1/S checkpoint. Downregulation of TYMS in vitro decreased cell proliferation and sensitized tumor cells to antimetabolite chemotherapy. In addition, depletion of TYMS in vivo by TYMS shRNA reduced tumor incidence, delayed tumor progression and prolonged survival in hTS/Ink4a/Arf−/− mice. Our data shows that activation of TYMS in Ink4a/Arf null background enhances uncontrolled cell proliferation and tumor growth, supporting the development of new agents and strategies targeting TYMS to delay tumorigenesis and prolong survival.

8-oxo-dGTP curbs tumor development via S phase arrest and AIF-mediated apoptosis

Oxidative stress can attack precursor nucleotides, resulting in nucleic acid damage in cells. It remains unclear how 8-oxo-dGTP and 8-oxoGTP, oxidized forms of dGTP and GTP, respectively, could affect DNA or RNA oxidation levels and tumor development. To address this, we intravenously administered 8-oxo-dGTP and 8-oxoGTP to wild-type and MTH1-knockout mice. 8-oxoGTP administration increased frequency of tumor incidence, which is more prominent in MTH1-knockout mice. However, 8-oxo-dGTP treatment rather reduced tumor development regardless of the mouse genotype. The tumor suppressive effects of 8-oxo-dGTP were further confirmed using xenograft and C57/6J-ApcMin/Nju mouse models. Mechanistically, 8-oxo-dGTP increased the 8-oxo-dG contents in DNA and DNA strand breakage, induced cell cycle arrest in S phase and apoptosis mediated by AIF, eventually leading to reduced tumor incidence. These results suggest distinct roles of 8-oxo-dGTP and 8-oxoGTP in tumor development.

Abstract P004: Targeting the RXR pathway for the prevention of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor (ER), progesterone receptor, and Erb-B2 receptor tyrosine kinase 2. TNBC patients exhibit a poor prognosis, even if treated with chemotherapy. Although studies using selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) have shown that breast cancer prevention is feasible, these drugs do not prevent ER-negative or TNBC tumors. It has been shown by our laboratory and others that retinoid X receptor (RXR)-specific ligands (rexinoids) can prevent breast cancers that are both ER-positive and ER-negative in mice. In our previous studies in MMTV-erbB2 mice, IRX-4204, a fourth generation rexinoid, prevented the development of most HER2/erbB2-positive tumors in these mice. For this study, we hypothesized that by targeting RXR pathway, we can prevent the development of triple negative, BRCA1-mutant mammary tumors in mice. To test the hypothesis, we treated MMTV-Cre/BRCA1co/co/p53+/- mice prior to their developing tumors with IRX-4204 to determine whether this RXR agonist effectively prevents triple-negative breast tumors. Methods: We tested the tumor preventative effect of IRX-4204 using the established MMTV-Cre/BRCA1co/co/p53+/- mouse model. These mice were produced by breeding MMTV-Cre/BRCA1co/co/p53+/- males and MMTV-Cre/BRCA1co/co/p53+/+ females, and female pups were PCR genotyped. All mice were separated into 4 groups: 1) sesame oil control, 2) IRX-4204 (10 mg/kg), 3) IRX-4204 (20 mg/kg) and 4) 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage, five days a week from 4 months of age. Mice were observed daily for tumor formation and toxicity. The percentage of tumor free mice were recorded, from which tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the Log-rank test. Results: In MMTV-Cre/BRCA1co/co/p53+/- mice, IRX-4204 reduced tumor incidence and was associated with an increase in median tumor free survival time from 209 days to 336 days at 10 mg/kg dose (p=0.005). At the higher dose (20 mg/kg) IRX-4204 also delayed tumor formation with median tumor free survival from 209 days to 260 days (p=0.039). The rexinoid 9-cis-UAB 30 also significantly delayed tumor formation in MMTV-Cre/BRCA1co/co/p53+/- mice with median survival from 209 days to 270 days (p=0.04). Long term treatment of IRX-4204 was not associated with any toxicity. Conclusion: RXR agonist IRX-4204 delayed ER-negative mammary tumor formation in BRCA1co/co; MMTV-Cre; p53+/- mice. Based on our results, IRX-4204 is an effective cancer preventive drug without observed toxicity. Our results suggest that studies with reduced IRX-4204 dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the IRX-4204 should be considered for the prevention of breast cancer in high-risk patients. (Supported by NCI-PREVENT contract to P. Brown and A. Mazumdar HHSN26100008). Citation Format: Cassandra Moyer, Jamal Hill, Darian Coleman, Shizuko Sei, Altaf Mohammed, Martin Sanders, Powel Brown, Abhijit Mazumdar. Targeting the RXR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P004.

Design of pH-responsive polymeric nanocarrier for targeted delivery of pyrogallol with enhanced antitumor potential in colon cancer

PurposeTo synthesize a polymeric pH-sensitive nanocarrier for the delivery of pyrogallol and investigate the anti-tumor activity of pyrogallol-loaded polymeric nanogel against colon cancer in rats. MethodsPoly(ethylene glycol)/polyacrylic acid (PEG/PAAc) nanogel was performed using gamma irradiation technique at irradiation doses; 30,40, and 50 kGy. The particle size distribution and diameter were investigated under the influence of various parameters by using dynamic light scattering analysis (DLS). The particle size was diminished by increasing AAc content and irradiation dose. Characterization of the performed nanogel was performed by (FT-IR) and (TEM). In vitro drug release behavior of the nanogel towards pyrogallol drug was assessed. Furthermore, the anti-cancer therapeutic efficiency of pyrogallol loaded PEG/PAAc nanogel was evaluated in a chemically induced colon cancer model in rats. ResultsPyrogallol/PEG/PAAc significantly reduced tumor incidence and volume as compared to DMH group. Also, it activated apoptotic pathway via up-regulating Bax, cytochrome C, cleaved caspase-3, p53, and down-regulating Bcl-2 expression. Furthermore, it attenuated cell cycle progression via reducing Cyclin A, Cyclin D1, and Cyclin E expression. It exhibited anti-proliferative activity through inhibiting PI3K/AKT signaling and downregulating the phosphorylation of AKT. It reduced pro-inflammatory cytokines TNF-α and IL-6. Results were confirmed by histopathological examination of colonic tissue. Interestingly, pyrogallol/PEG/PAAc demonstrated anti-tumor potential more efficiently than free pyrogallol, revealing localized drug delivery. ConclusionThis formulation could be considered as a promising agent in the treatment of colon cancer.

Abstract 940: Loss of Pkd1 limits colorectal cancer by increasing colon barrier function

Abstract Colorectal cancer (CRC) is one of the most common cancers, with an annual incidence of ~135,000 in the US, associated with ~ 50,000 deaths. CRC typically arises from mutations that inactivate the tumor suppressor APC, which activates the WNT/CTNNB1 pathway to drive cell transformation and tumor growth. Identifying factors that reduce incidence of CRC is of high interest. Intriguingly, a large population study has found a decreased incidence of CRC in patients with autosomal dominant polycystic kidney disease (ADPKD), an inherited disease affecting approximately 1 in 1,000 individuals. ADPKD typically arises from germline mutations in the PKD1 gene, which encodes a transmembrane signaling protein that is ubiquitously expressed. Puzzlingly, activated WNT/CTNNB1 signaling is also found in ADPKD, which would be expected to enhance rather than reduce likelihood of CRC. To investigate the role of PKD1 in modulating CRC tumorigenesis, we established a mouse model with tamoxifen-inducible loss of Apc with or without parallel loss of Pkd1 in the colon. Pkd1 loss vey significantly reduced tumor incidence (~80%) and rate of growth. In addition, growth of Pkd1-/-;Apc-/- organoids was reduced relative to Apc-/-organoids, indicating a cancer cell-intrinsic activity. Analysis of signaling pathway activation by RNA-seq indicated that Pkd1-deficient CRC organoids had enhanced WNT/CTNNB1 signaling, excluding reduced activation of this core driver pathway as a mechanism of action. Considering alternative mechanisms, a prominent feature of ADPKD is reprogramming barrier function in renal cells to reduce permeability, supporting cyst development; notably, compromised integrity of the epithelial barrier is associated with CRC. We tested the hypothesis that loss of Pkd1 increased colon barrier integrity. As a first approach, we compared susceptibility of wt versus Pkd1-/- mice to dextran-sodium sulfate (DSS), which induces colitis by damaging epithelial barriers. Histopathological assessment confirmed significantly less DSS-induced damage of colon tissue in Pkd1-/- mice. Further, treatment of DSS-treated mice with orally gavaged FITC-dextran led to elevated FITC-dextran in the serum of wt versus Pkd1-/- mice, indicating greater passage through a functionally impaired barrier. In ADPKD, decreased barrier permeability is mediated by upregulation of specific claudins (CLDN4, 7); in CRC, these claudins are typically reduced during and promote tumor formation. We detected strong elevation of CLDN4 and CLDN7 in the colonic epithelium of Pkd1-/- versus wt mice, with the expressed proteins having consistently greater localization to cell junctions. Based on these and other data, we identify PKD1 control of barrier function as an important regulatory mechanism in CRC tumorigenesis, which may suggest novel therapeutic strategies based on targeting PKD1-associated effector pathways. Citation Format: Anna S. Nikonova, Alexander Y. Deneka, Flaviane Silva, Rosella Tricarico, Anna Kiseleva, Sergey Grivennikov, Erica A. Golemis. Loss of Pkd1 limits colorectal cancer by increasing colon barrier function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 940.

Abstract 5954: Dietary sugar promotes breast tumorigenesis partially through upregulating 12 lipoxygenase/PPARd signaling and remodeling breast tumor microenvironment

Abstract Ample evidence from epidemiological studies links high-sugar diets with increased breast cancer risk, but the underlying molecular mechanisms remain unclear. We previously reported that sucrose-enriched diets (SED) accelerated breast tumorigenesis in MMTV-ErbB2 mice and promoted the breast tumor growth in orthotopic models of mouse (4T1) or human breast cancer cells (MDA-MB-231) by up-regulating 12-lipoxygenase (12-LOX) and its arachidonate metabolite 12-HETE. In this report, we performed mechanistic studies to determine the role of 12-LOX in SED-induced breast tumorigenesis and also examined the SED effects on remodeling breast tumor microenvironment during SED-induced breast tumorigenesis. We found SED (diet with 125 g/kg sucrose), at a concentration equivalent to the average sugar consumption of American population, significantly increased the 4T1 orthotopic tumor weights by 3.7-fold in average compared to the isocaloric cornstarch control diet group. We obtained data showing that 12-LOX downregulation by CRISPR/Cas9 technology not only significantly reduced tumor incidence and inhibited the tumor growth, but also blocked the SED-promoted breast tumor growth in the MDA-MB-231 cell mouse orthotopic model. We also found that SED increased expression of peroxisome proliferator-activated receptor-delta (PPARd), a lipid nuclear receptor along with its target genes (i. g., LPL, CD36 and SCD1) in 4T1 orthotopic breast tumor tissues. 12-LOX downregulation decreased PPARD expression in MDA-MB-231 cells, while 12-HETE increased PPARd and its target proteins such as PDK4 and ANGPLT4 expression levels in 4T1 cells. Together, these data suggest PPARd as a downstream target gene of 12-LOX. Additionally, our studies showed that SED significantly increased chemokine CCL2 in 4T1 orthotopic tumor tissues and 12-HETE led to higher secreted CCL2 in 4T1 mouse breast cancer cells which was blocked by suppressing PPARd expression. Furthermore, we observed 10-fold higher CD11b+/Ly6G+ cells in SED-induced 4T1 tumor tissues by flow cytometry. Among CD45+ cells, percentage of CD8+ T cells was decreased by 34% in SED-induced 4T1 tumors, and percentages of CD8+/CD69+ (active CD8+ T cells) and CD8+/GrzB+ (effector T cells) were reduced by 78% and 60%, respectively, in SED-induced 4T1 tumors, suggesting SED also led to immune suppressive tumor microenvironment (iTME) during breast tumorigenesis. We further noted that MDSCs, but not macrophages, expressed higher CCR2 in TILs of SED fed tumor than that of cornstarch control diet group, indicating SED induced accumulation of MDSCs is likely mediated by CCL2. In conclusion, our data strongly supports added sugar (sucrose) to accelerate the development and progression of breast cancer, potentially involving up-regulation of the expression of 12-LOX and PPARd as well as iTME. Citation Format: Sharmistha Chakraborty, Xiangsheng Zuo, Venkatesh Hedge, Megan Tran, Yan Jiang, Mihai Gagea, Jagannadha Sastry, Peiying Yang. Dietary sugar promotes breast tumorigenesis partially through upregulating 12 lipoxygenase/PPARd signaling and remodeling breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5954.

Long-Term Supplementation with Fruits and Vegetables Prolongs Lifespan and Reduces Tumor Incidence in Mice Fed a Western-Style High-Fat Diet

ObjectivesEpidemiological studies suggest that consumption of fruits and vegetables (FV) was negatively associated with the incidence of certain cancers and positively associated with life expectancy. To date, a causal relationship has not been demonstrated. The objectives of the current study were to investigate the effects of long-term FV supplementation on health- and life-span in normal weight and obese mice. MethodsMale C57BL/6J mice (5-wk) were randomly assigned to one of four groups (60/group): low fat control (LF-C, 10% kcal fat), high fat control (HF-C, 45% kcal fat), and each with 15% of a unique mixture of FV (patent pending) (w/w) (LF-FV and HF-FV). All mice were euthanized when a group reached 50% mortality. Survival analysis was performed using log-rank (Mantel-Cox) test; a two-sided Fisher’s exact test was performed to compare difference in tumor incidence. ResultsAfter 21 months of feeding, HF-C group was the first to reach 50% mortality. Further, a Kaplan-Meier survival curve demonstrated that, at termination, HF-C group had higher mortality (50.0%) compared to LF-C group (18.3%, p = 0.0008). Notably, HF-FV group had significantly lower mortality (23.3%) compared to HF-C mice (p = 0.008), and there was no significant difference in mortality between HF-FV and LF-C. Mortality was lower in LF-FV (11.7%) compared to LF-C (18.3%), although this difference was not statistically significant. Furthermore, tumor incidence in HF-C group (73.3%) was significantly higher than that in LF-C group (30.0%, p < 0.0001). HF-FV group had 23.3% lower tumor incidence compared to HF-C group (p = 0.014). There was no significant difference in tumor incidence between LF-C (30.0%) and LF-FV groups (31.7%). ConclusionsOur findings provide the first causal evidence that long-term intake of a diet supplemented with a wide variety of fruits and vegetables could extend lifespan and decrease tumor incidence in mice fed a Western-style high-fat diet. These results provide a foundation for further investigation into the benefits of fruit and vegetable supplementation on aging and age-related disease. Funding SourcesThis material is based upon work supported by the U.S. Department of Agriculture – Agricultural Research Service (ARS), under Agreement No. 58–8050-9–004.

C-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity

SummaryMetabolic syndrome is associated with obesity, insulin resistance, and the risk of cancer. We tested whether oncogenic transcription factor c-JUN metabolically reprogrammed cells to induce obesity and cancer by reduction of glucose uptake, with promotion of the stemness phenotype leading to malignant transformation. Liquid alcohol, high-cholesterol, fat diet (HCFD), and isocaloric dextrin were fed to wild-type or experimental mice for 12 months to promote hepatocellular carcinoma (HCC). We demonstrated 40% of mice developed liver tumors after chronic HCFD feeding. Disruption of liver-specific c-Jun reduced tumor incidence 4-fold and improved insulin sensitivity. Overexpression of c-JUN downregulated RICTOR transcription, leading to inhibition of the mTORC2/AKT and glycolysis pathways. c-JUN inhibited GLUT1, 2, and 3 transactivation to suppress glucose uptake. Silencing of RICTOR or c-JUN overexpression promoted self-renewal ability. Taken together, c-JUN inhibited mTORC2 via RICTOR downregulation and inhibited glucose uptake via downregulation of glucose intake, leading to self-renewal and obesity.

Effects of Hibiscus sabdariffa Calyxes Aqueous Extract on Antioxidant Status and Histopathology in Mammary Tumor-Induced in Rats.

Breast cancer is a major threat worldwide. Hibiscus sabdariffa is widely consumed as beverage in sub-Saharan Africa for its anticancer potential. The present study therefore aimed at scientifically verifying its anticancer effect in rats. For this, 48 Wistar rats (∼55 days) were treated either with tamoxifen at 3.3 mg/kg BW (standard) or with a decoction of H. sabdariffa (125, 250, and 500 mg/kg BW) or distilled water (vehicle). Breast cancer was induced by a single dose of 50 mg/kg of 7,12-dimethylbenz(a)anthracene (DMBA). At the end of the 21 weeks of treatment, the tumor incidence, tumor morphology, histopathology, as well as some biochemical parameters in the tumors were assessed. As a result, 86% of DMBA's rats developed mammary tumors. The H. sabdariffa extract (125 and 250 mg/kg) reduced tumor incidence by 63% and 75%, respectively; inhibited tumor burden by 84.86% and 38.78%, respectively, and decreased tumor volume by more than 72% compared to the DMBA group. It also protected rats against DMBA-induced diffuse breast neoplasia, and the optimal effect was recorded at 125 mg/kg. Furthermore, it significantly increases the SOD activity and decreases the MDA level. In summary, H. sabdariffa has antibreast tumor and antioxidant properties in rats, which could justify its common use to treat cancer.

A tumor suppressor role for EZH2 in diffuse midline glioma pathogenesis

Pediatric high-grade gliomas, specifically diffuse midline gliomas, account for only 20% of clinical cases but are 100% fatal. A majority of the DMG cases are characterized by the signature K27M mutation in histone H3. The H3K27M mutation opposes the function of enhancer of zeste homolog 2 (EZH2), the methyltransferase enzyme of the polycomb repressor complex 2. However, the role of EZH2 in DMG pathogenesis is unclear. In this study, we demonstrate a tumor suppressor function for EZH2 using Ezh2 loss- and gain-of-function studies in H3WT DMG mouse models. Genetic ablation of Ezh2 increased cell proliferation and tumor grade while expression of an Ezh2 gain-of-function mutation significantly reduced tumor incidence and increased tumor latency. Transcriptomic analysis revealed that Ezh2 deletion upregulates an inflammatory response with upregulation of immunoproteasome genes such as Psmb8, Psmb9, and Psmb10. Ezh2 gain-of-function resulted in enrichment of the oxidative phosphorylation/mitochondrial metabolic pathway namely the isocitrate dehydrogenase Idh1/2/3 genes. Pharmacological inhibition of EZH2 augmented neural progenitor cell proliferation, supporting the tumor suppressive role of EZH2. In vivo 7-day treatment of H3K27M DMG tumor bearing mice with an EZH2 inhibitor, Tazemetostat, did not alter proliferation or significantly impact survival. Together our results suggest that EZH2 has a tumor suppressor function in DMG and warrants caution in clinical translation of EZH2 inhibitors to treat patients with DMG.