Louisiana Research Day Program Book 2025

Biomedical Research: Section 2

Biomedical Research: Section 2

Md Ashiq Mahmud, MSc 1 ; Abu Bakar Siddique, PhD 1 ; Afsana Tajmim, PhD 1 ; Judy Ann King, PhD 2 ; Khalid A El Sayed, PhD 1 ; 1 University of Louisiana at Monroe, College of Pharmacy, Monroe, LA; 2 Foundational and Clinical Sciences Department, Thomas F. Frist, Jr. College of Medicine, Belmont University, Nashville, TN 25 THE OLIVE OIL MONOPHENOLIC SECOIRIDOID LIGSTROSIDE AGLYCONE SUPPRESSES MELANOMA PROGRESSION BY TARGETING THE BRAF SIGNALING PATHWAY

Md Saqline Mostaq, MS; Md Nurul Amin, MS; Amanda Raphael, P4; Davorka Sekuli ć, MD ; Lin Kang, PhD; Pawel Michalak, PhD; Yong-Yu Liu, PhD University of Louisiana at Monroe 26 INHIBITION OF GLUCOSYLCERAMIDE SYNTHASE REACTIVATES P53-SUPPRESSIVE EFFECTS ON CANCER STEM CELLS THROUGH M6A MODIFICATION AT P53 R273 MUTANT CODON

Context: Melanoma is among the most abundant malignancies in the US and worldwide. Ligstroside aglycone (LA) is a rare extra-virgin olive oil-derived monophenolic secoiridoid with diverse bioactivities. Objective: Study aims to evaluate the therapeutic potential of Ligstroside aglycone (LA) as a novel treatment for skin cancer. Methods: LA dose–response screening at the NCI 60 cancer cells panel identified the high sensitivity of the Malme-3M cell line, which harbors a BRAF V600E mutation. Daily oral 10 mg/kg LA exhibited potent in vivo antitumor effects against Malme-3M cells xenograft in a nude mouse model by targeting the BRAF signaling pathway. Results: A human Clariom S microarray analysis of the collected Malme- 3M tumors identified 571 dysregulated genes, with the downregulation of pathways critical for melanoma cells growth and survival. A Western blot analysis of the collected animal tumors further validated the downregulation of the mutated BRAF–MAPK axis, as well as

the GPD1 and ELOVL6 expression levels. A histopathological analysis of Malme-3M tumor sections showed extensive focal tumor necrosis in treated mice. An immunofluorescence study of tumor sections showed notable reductions in proliferation marker ki67 and the vasculogenesis marker CD31 in treated tumors. Conclusion: These findings promote LA as a potential nutraceutical lead for the control of the BRAF V600E mutant melanoma.

Background: The tumor suppressor protein p53, encoded by TP53, plays a pivotal role in preventing tumorigenesis. However, missense mutations of TP53, prevalent in over 50% of cancers, drive drug resistance and tumor metastasis. Glucosylceramide synthase (GCS), encoded by UGCG, catalyzes ceramide glycosylation, promoting drug resistance and cancer progression. GCS overexpression, often induced during chemotherapy, exacerbates pharmacological strategies can reverse drug resistance in colon cancer cells with the p53 R273H+/+ mutation. CRISPR/Cas9-mediated UGCG knockout (UGCG-KO) or treatment with the GCS inhibitor GENZ-667161 (Genz-161) sensitized WiDr cells to oxaliplatin, irinotecan, and paclitaxel. UGCG-KO cells exhibited a 2-fold lower IC50 for oxaliplatin and irinotecan, as well as a 4-fold reduction in cell migration. Tumor volume reductions of 63% and 69% were observed with oxaliplatin in combination with UGCG-KO or Genz-161, respectively, compared to oxaliplatin alone. Lipidomics revealed decreased ceramide glycosylation, with N16 glycosphingolipid implicated in drug these effects. This study demonstrates that targeting GCS using genetic and

resistance. Single-nucleus RNA sequencing (snRNA-seq) showed an 89% reduction in stem-like cell populations in UGCG-KO tumors, confirmed by immunohistochemistry, which also indicated increased ceramide and p53 expression alongside reduced cancer stem cell populations. Imaging flow cytometry revealed that combination treatment with Genz-161 and irinotecan reduced cancer stem cell (CSC) populations to 4%, compared to 8.5% in Irinotecan treated cells. Similarly, UGCG-KO cells treated with irinotecan alone showed a CSC population of 4.2%. Western blot analysis demonstrated increased phosphorylated p53 and p21 expression, alongside decreased MDR1 expression, in cells treated with irinotecan and Genz-161 or UGCG-KO cells treated with irinotecan alone. Additionally, we uncovered a novel role of m6A RNA modification in GCS mediated drug resistance. Inhibiting ceramide glycosylation reduced METTL3 expression and m6A modification at the mutant p53 R273H codon, thereby downregulating mutant p53 via YTHDF2. Neplanocin A (NPC), an m6A RNA methylation inhibitor, effectively reversed drug resistance in cellular and tumor models. Our findings reveal the critical interplay

between ceramide glycosylation and m6A RNA methylation in cancer drug resistance. Targeting GCS to disrupt m6A modifications at mutant p53 codons offers a promising therapeutic strategy to overcome chemoresistance and eliminate cancer stem cells in p53-mutated tumors.

36

37

2025 Research Recognition Day