Bhaskar V. S. Kallakury

Approximately 50% of individuals diagnosed with Stage I liver cancer live beyond four years; however, a small subset of Stage I patients die within the first year. A prognostic biomarker panel that can identify high-risk Stage I patients may be extremely valuable. In this study, we used the Long–Evans Cinnamon (LEC) rat model of Wilson’s Disease and hepatocellular carcinoma (HCC), along with data from The Cancer Genome Atlas (TCGA) human database, to create a novel biomarker panel. We generated and analyzed a rat microarray gene expression profile by comparing liver tumor tissues with adjacent normal tissues from the same animals, covering approximately 30,000 genes. The microarray results were translated into a five-gene panel associated with 1-year survival in Stage I liver cancer patients based on TCGA data, in combination with machine learning and bioinformatics approaches. The panel was internally validated following the “REporting recommendations for Tumor MARKer prognostic studies (REMARK)” guidelines. With no existing Stage-I-specific prognostic tools, a biomarker panel associated with 1-year survival in patients with Stage I liver cancer is a potential candidate for rigorous external validation.

Long-duration space missions expose astronauts to galactic cosmic radiation (GCR), a complex spectrum of high-charge, high-energy (HZE) ions that pose significant risks of chronic tissue injury. To model these effects, we examined intestinal outcomes in wild-type mice 5 months after low-dose (50 cGy) 33-ion mixed-field GCR simulation (GCRsim). GCRsim induced sustained DNA double-strand breaks (DSBs) and oxidative stress, as shown by elevated γH2AX foci and 4-HNE staining. Intestinal epithelial cells (IECs) exhibited pronounced senescence, marked by increased SA-β-gal activity, p16 upregulation, LaminB1 loss, and induction of senescence-associated secretory phenotype (SASP) cytokines (Cxcl10, IL-6, IL-1β, Icam1). GCRsim also elevated circulating LINE-1 DNA and reduced expression of DNA-degrading nucleases (DNase2, TREX1), indicating impaired extracellular DNA clearance. Targeted molecular study revealed persistent activation of the cGAS–STING pathway, with elevated cGAS, STING, pTBK1, pIKKα/β, and nuclear pIRF3, pIRF7, and p65, consistent with chronic innate immune signaling. Functionally, GCRsim altered nutrient absorption gene expression—upregulating glucose transporters (Slc2a2, Slc2a5, Slc5a1) and gut hormones (Cck, Gip), while downregulating cholesterol/fat transporters (Npc1, Npc1l1). Biochemical markers supported intestinal injury, with decreased serum citrulline and increased intestinal fatty acid-binding protein (I-FABP), indicating barrier compromise. Collectively, these findings demonstrate that GCRsim drives sustained intestinal dysfunction, highlighting the need for countermeasures to protect GI health during deep-space missions.

Heavy ion radiation, prevalent in outer space and relevant for radiotherapy, is densely ionizing and poses a risk to intestinal stem cells (ISCs), which are vital for maintaining intestinal homeostasis. Earlier studies have shown that heavy-ion radiation can cause chronic oxidative stress, persistent DNA damage, cellular senescence, and the development of a senescence-associated secretory phenotype (SASP) in mouse intestinal mucosa. However, the specific impact on different cell types, particularly Lgr5⁺ intestinal stem cells (ISCs), which are crucial for maintaining cellular homeostasis, GI function, and tumor initiation under genomic stress, remains understudied. Using an ISCs-relevant mouse model (Lgr5⁺ mice) and its GI tumor surrogate (Lgr5⁺Apc^1638N/+ mice), we investigated ISCs-specific molecular alterations after high-LET radiation exposure. Tissue sections were assessed for senescence and SASP signaling at 2, 5 and 12 months post-exposure. Lgr5+ cells exhibited significantly greater oxidative stress following ²⁸Si irradiation compared to γ-ray or controls. Both Lgr5⁺ cells and Paneth cells showed signs of senescence and developed a senescence-associated secretory phenotype (SASP) after ²⁸Si exposure. Moreover, gene expression of pro-inflammatory and pro-growth SASP factors remained persistently elevated for up to a year post-²⁸Si irradiation. Additionally, p38 MAPK and NF-κB signaling pathways, which are critical for stress responses and inflammation, were also upregulated after ²⁸Si radiation. Transcripts involved in nutrient absorption and barrier function were also altered following irradiation. In Lgr5⁺Apc^1638N/+ mice, tumor incidence was significantly higher in those exposed to ²⁸Si radiation compared to the spontaneous tumorigenesis observed in control mice. Our results indicate that high-LET ²⁸Si exposure induces persistent DNA damage, oxidative stress, senescence, and SASP in Lgr5⁺ ISCs, potentially predisposing astronauts to altered nutrient absorption, barrier function, and GI carcinogenesis during and after a long-duration outer space mission.

Estimation of cancer risk among astronauts planning to undertake future deep-space missions requires understanding the quantitative and qualitative differences in radiogenic cancers after low- and high-LET radiation exposures. Previously, we reported a multifold higher RBE for high-LET radiation-induced gastrointestinal (GI) tumorigenesis in Apc^1638N/+ mice. Using the same model system, i.e., Apc^1638N/+ mice, here, we report qualitative differences in the cellular phenotype of low- and high-LET radiation-induced GI tumors. Stem cell (SC) phenotypes were identified using BMI1, ALDH1, CD133, DCLK1, MSI1, and LGR5 markers in low (γ-rays)- and high (⁵⁶Fe)-LET radiation-induced and spontaneous tumors. We also assessed the expression of these markers in the adjacent normal mucosa. All six of these putative SC markers were shown to be overexpressed in tumors compared to the adjacent normal intestinal tissue. A differential SC phenotype for spontaneous and radiogenic intestinal tumors in Apc^1638N/+ mice was observed, where the ALDH1, BMI1, CD133, MSI1, and DCLK1 expressing cells were increased, while LGR5 expressing cells were decreased in ⁵⁶Fe-induced tumors compared to γ-ray-induced and spontaneous tumors. Furthermore, higher β-catenin activation (marked by nuclear localization) was observed in ⁵⁶Fe-induced tumors compared to γ and spontaneous tumors. Since differential tumor cell phenotype along with activated β-catenin may very well affect malignant progression, our findings are relevant to understanding the higher carcinogenic risk of high-LET radiation. This study has implications for the assessment of GI-cancer risk among astronauts, as well as for the estimation of secondary cancer risk among patients receiving hadron therapy, considering that our results indicate increased stemness properties after radiation.

MiR-150-5p is frequently deregulated in cancer, with expression and mode of action varying according to the tumor type. Here, we investigated the expression levels and role of miR-150-5p in the aggressive breast cancer subtype triple-negative breast cancer (TNBC). MiR-150-5p expression levels were analyzed in tissue samples from 113 patients with invasive breast cancer (56 TNBC and 57 non-TNBC) and 41 adjacent non-tumor tissues (ANT). Overexpression of miR-150-5p was observed in tumor tissues compared with ANT tissues and in TNBC compared with non-TNBC tissues. MiR-150-5p expression levels were significantly associated with high tumor grades and the Caucasian ethnicity. Interestingly, high miR-150-5p levels were associated with prolonged overall survival. Manipulation of miR-150-5p expression in TNBC cells modulated cell proliferation, clonogenicity, migration, and drug resistance. Manipulation of miR-150-5p expression also resulted in altered expression of its mRNA targets, including epithelial-to-mesenchymal transition markers, MYB, and members of the SRC pathway. These findings suggest that miR-150-5p is overexpressed in TNBC and contributes to the aggressiveness of TNBC cells in vitro.

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients.

As current interventions for chronic hepatitis B (CHB) rarely induce cure, more effective drugs are needed. Short-term treatment of woodchucks with the novel immunomodulator AIC649, a parapoxvirus-based stimulator of toll-like receptor 9 dependent and independent pathways, has been shown to reduce viral DNA and surface antigen via a unique, biphasic response pattern. The present study evaluated long-term AIC649 treatment in combination with Entecavir for potency and safety in woodchucks. AIC649 monotreatment induced modest reductions in serum viral DNA and surface and e antigens that were associated with the same biphasic response pattern previously observed. Entecavir monotreatment reduced transiently viremia but not antigenemia, while AIC649/Entecavir combination treatment mediated superior viral control. Undetectability of viral antigens and elicitation of antibodies in AIC649/Entecavir-treated woodchucks correlated with the expression of interferons and suppression of viral replication in liver. Combination treatment was well tolerated, and liver enzyme elevations were minor and transient. It was concluded that the AIC649-mediated effects were most likely based on an improvement and/or reconstitution of antiviral immune responses that are typically deficient in CHB. As a combination partner to Entecavir, the antiviral efficacy of AIC649 was markedly enhanced. This preclinical study supports future evaluation of AIC649 for treatment of human CHB.