In cancer patients, chronic paclitaxel (PTX) treatment causes excruciating pain, limiting its use in cancer chemotherapy. The neuroprotective potential of synthetic cannabidiol (CBD) and CBD formulated in extracellular vesicles (CBD-EVs) isolated from human umbilical cord derived mesenchymal stem cells was investigated in C57BL/6J mice with PTX-induced neuropathic pain (PIPN). The particle size of EVs and CBD-EVs, surface roughness, nanomechanical properties, stability, and release studies were all investigated. To develop neuropathy in mice, PTX (8 mg/kg, i.p.) was administered every other day (four doses). In terms of decreasing mechanical and thermal hypersensitivity, CBD-EVs treatment was superior to EVs treatment or CBD treatment alone (p < 0.001). CBD and CBD-EVs significantly reduced mitochondrial dysfunction in dorsal root ganglions and spinal homogenates of PTX-treated animals by modulating the AMPK pathway (p < 0.001). Studies inhibiting the AMPK and 5HT1A receptors found that CBD did not influence the neurobehavioral or mitochondrial function of PIPN. Based on these results, we hypothesize that CBD and CBD-EVs mitigated PIPN by modulating AMPK and mitochondrial function.

As breast cancer cells transition from letrozole-sensitive to letrozole-resistant, they over-express epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and human epidermal growth factor receptor 2 (HER2) while acquiring enhanced motility and epithelial-to-mesenchymal transition (EMT)-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Interestingly, glyceollin inhibits the proliferation and tumor progression of triple-negative breast cancer (TNBC) and estrogen-independent breast cancer cells; however, it is unlikely that a single phytochemical would effectively target aromatase-inhibitor (AI)-resistant metastatic breast cancer in the clinical setting. Since our previous report indicated that the combination of lapatinib and glyceollin induced apoptosis in hormone-dependent AI-resistant breast cancer cells, we hypothesized that combination therapy would also be beneficial for hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by decreasing the expression of proteins associated with proliferation and cell cycle progression. While glyceollin + lapatinib treatment caused comparable inhibitory effects on the proliferation and migration in both cell lines, combination treatment selectively induced S and G2/M phase cell cycle arrest of the LTLT-Ca cells, which was mediated by decreased cyclin B1. This phenomenon may represent a unique opportunity to design novel combinatorial therapeutic approaches to target hormone-refractory breast tumors.

Midasin (MDN1) is a chaperone protein required for maturation and nuclear export of pre-ribosomal RNA. Recent investigation regarding the mutational status of MDN1 in breast cancer (BC) revealed an increased tumor mutational burden in BC patients. Interestingly, our previously published work demonstrated significantly high MDN1 protein levels in AI-resistant mammospheres. Therefore, we investigated the overall status of MDN1 in the TCGA database in breast cancer tumors and identified MDN1 expression in breast cancer cells that were sensitive or resistant to AIs. The purpose of this study was to evaluate whether there is an association between MDN1 expression or mutation status to examine if MDN1 functions as an early high-risk indicator for breast cancers resistant to AIs or endocrine therapy. Using the cBioportal for Cancer Genomics, in silico analysis was conducted and BC patients with altered MDN1 status demonstrated several clinical attributes that were significantly associated with higher mutation counts (Kruskal Wallis test, p value = 1.41 × 10-10 and q value = 4.11 × 10-9); MSI sensor score, (p value = 2.74 × 10-5 and q value = 2.27 × 10-4); and Fraction Altered Genome score (p value = 2.37 × 10-3 and q value = 0.011). Next, aneuploidy in breast cancer significantly evolved and was positively correlated with higher MDN1 expression (Spearman: 0.64, p value = 3.92 × 10-122). Compared to the other BC subtypes, MDN1 was significantly elevated in basal breast cancer (Chi square test p value &lt; 10.0 × 10-10, q value = 4.11 × 10-9) and significantly higher in deceased versus living BC patients. The DepMap breast cancer cell line database analysis also revealed significantly increased MDN1 expression in both ER+/HER2- cell lines (p value = 7.93 × 10-3; 12 cell lines) and in ER-/HER2- cell lines (p value = 2.88 × 10-2; 31 cell lines). Primary and metastatic BC cell lines were compared and MDN1 expression was significantly higher in metastatic BC cells (p value = 8.04 × 10-5; 33 metastatic cell lines). Likewise, AI-resistant cell lines exhibited altered MDN1 expression compared to their sensitive counterparts. Additionally, there was a statistically significant association between higher ER copy number and increased MDN1 expression in BC cell lines (p value = 1.44 × 10-3; 61 cell lines). Further in-depth analysis of proteins that were coexpressed in MDN1-altered tumors revealed a panel of genes (MSH2, MSH6, RBM15, FOXM1,CCNE2, and CHEK1) that could represent a signature of early markers of AI-resistant BC since there was a positive correlation between MDN1 expression and associated pathways. In conclusion, increased expression of MDN1 in BC tumors or cells may serve as preliminary marker to predict resistance to endocrine therapy due to its significant association with genes such as MSH2, MSH6 and FOXM1 that are strongly established as indicators of endocrine resistance. This work was supported by NIH grant # SC1GM126617 and U54MD007582. Citation Format: Afia Ohemeng, Akash Gupta, Bipika Banjara, Mounika M. Pamukuntla, Manasa Kotina, A Michael Davidson, Syreeta L. Tilghman. MDN1, a novel potential early biomarker for endocrine resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2051.

As breast cancer cells transition from letrozole-sensitive to letrozole resistance they over-express EGFR, MAPK, and HER2 and acquire enhanced motility and EMT-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Since previous reports from our lab demonstrate that the combination of lapatinib, a dual EGFR and HER2 inhibitor, and glyceollin, a novel phytochemical, induce apoptosis in hormone-dependent aromatase inhibitor (AI)-resistant breast cancer cells, we hypothesized that combination therapy could reverse motility in hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by altering proteins involved in motility and cell cycle progression. We compared the effects of 10 μM glyceollin ± 5 μM lapatinib treatment on both cell lines and evaluated cell viability, cell migration, cell cycle analysis, and protein expression of cell cycle regulators. Glyceollin ± lapatinib treatment caused a time-dependent decrease in cell viability which was comparable between each cell line. When wound healing assays were conducted, glyceollin prevented the migration in the AC-1 and LTLT-Ca cell lines by approximately 55% and 51% respectively, while combination treatment exhibited synergistic inhibitory properties by preventing 88% and 93% wound closure in the AC-1 cells and LTLT-Ca cells, respectively. To determine if the glyceollin + lapatinib-induced decrease in cell viability was a consequence of cell cycle dysregulation, flow cytometric analyses were performed and drug treatment had no effect on the AC-1 cell cycle distribution however, combination therapy reduced the number of LTLT-Ca cells in the G1/G0 phase, while causing accumulation of the cells in both the S phase and G2/M phase. Since combination therapy altered the cell cycle distribution of the LTLT-Ca, we measured the effect of glyceollin ± lapatinib treatment on the expression of key cell cycle regulators such as p21, p27, cyclin B1, cdk1, and cdk2. Interestingly, while there there was no effect on protein expression in the AC-1 cells, glyceollin ± lapatinib treatment selectively decreased the expression of cyclin B1 in the LTLT-Ca cells, without significantly altering the expression of the other proteins. These results suggest that while both AI-sensitive and AI-resistant cells respond favorably to the growth inhibitory and anti-migratory properties of glyceollin + lapatinib, the LTLT-Ca cells exhibit increased sensitivity to cell cycle progression, which may represent an opportunity to exploit and refine second-line therapies for AI-resistant cancers. This work was supported by NIH grant #1SC1GM126617 and U54MD007582. Citation Format: Bipika Banjara, Jankiben R. Patel, A Michael Davidson, Syreeta L. Tilghman. Novel therapeutic combination targets the growth of letrozole resistant breast cancer through decreased cyclin B1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 406.