(W1030-05-28) Effects of B4GALNT1 Expression on Metastatic Phenotype and Response to Treatment in Osteosarcoma Cell Lines

Purpose: Osteosarcoma is the most common type of bone tumor that has the highest prevalence in adolescents and young adults. Currently, the standard protocol for osteosarcoma treatment consists of surgery and chemotherapy, but despite the progress that has been made in previous decades, still 30-50% of patients experience recurrence. GD2 is a glycosphingolipid and a member of the ganglioside family that is overexpressed in several tumor tissues such as osteosarcoma and neuroblastoma while having limited expression in most normal tissues.This glycosphingolipid is synthesized by an enzyme called GD2 synthase that is coded by the B4GALNT1 gene. Recent studies have shown that the expression of B4GALNT1 in cancer cells can be related to a more malignant phenotype. In this study, we sought to understand the effects of B4GALNT1 expression on cancer cells’ behavior and whether the expression of this gene can cause more metastatic phenotype and resistance to chemotherapy.

Methods: In order to confirm the accuracy of our results and conclusions, all steps were performed in two different OS cell lines, HOS and SAOS. We used B4GALNT1 plasmid for overexpression and CRISPR/CAS9 for knocking out the desired gene. The changes in the expression were confirmed using RT-qPCR, comparing them to wild type cell lines. To evaluate the metastatic phenotype we first compared the expression of epithelial and mesenchymal markers (E-cadherin and N-cadherin) and transcription factors for epithelial to mesenchymal transition (Twist, Snail, and Slug). In the next step, the migration capability of Knocked out, over-expressed and wild type cell lines was compared using 24 hour wound healing assay. In the last step of the experiment, the sensitivity and resistance of each three types of each osteosarcoma cell line were tested using Cisplatin, Methotrexate, and Doxorubicin.

Results: There were considerable differences in terms of metastatic markers (n-cadehrin, snail, slug) between cell with over expressed gene and knocked out cells. Also differences in migration time were observed in this cells.

Conclusion: Our results show that B4GALNT1 has considerable effects on metastatic phenotype and response of the cell lines to the standard protocol of treatment. This study shows that B4GALNT1 can be a potential gene to target in the treatment of metastatic osteosarcoma.

References: 1. Cancer statistics, 2018 - Siegel - 2018 - CA: A Cancer Journal for Clinicians - Wiley Online Library [Internet]. [cited 2022 Apr 26]. Available from: https://acsjournals.onlinelibrary.wiley.com/doi/full/10.3322/caac.21442
2. Sakamoto A, Iwamoto Y. Current Status and Perspectives Regarding the Treatment of Osteosarcoma: Chemotherapy. Reviews on Recent Clinical Trials. 3(3):228–31.
3. Thanindratarn P, Dean DC, Nelson SD, Hornicek FJ, Duan Z. Advances in immune checkpoint inhibitors for bone sarcoma therapy. Journal of Bone Oncology. 2019 Apr 1;15:100221.
4. miR‐134 inhibits osteosarcoma cell invasion and metastasis through targeting MMP1 and MMP3 in vitro and in vivo - Chen - 2019 - FEBS Letters - Wiley Online Library [Internet]. [cited 2022 Apr 26]. Available from: https://febs.onlinelibrary.wiley.com/doi/10.1002/1873-3468.13387
5. Berhe S, Danzer E, Meyers PA, Behr G, LaQuaglia MP, Price AP. Unusual abdominal metastases in osteosarcoma. Journal of Pediatric Surgery Case Reports. 2018 Jan 1;28:13–6.
7. Miwa S, Shirai T, Yamamoto N, Hayashi K, Takeuchi A, Igarashi K, et al. Current and Emerging Targets in Immunotherapy for Osteosarcoma. J Oncol. 2019 Jan 1;2019:7035045.
8. Jiang T, Wu H, Lin M, Yin J, Tan L, Ruan Y, et al. B4GALNT1 promotes progression and metastasis in lung adenocarcinoma through JNK/c-Jun/Slug pathway. Carcinogenesis. 2021 Apr 1;42(4):621–30.

Acknowledgments: N/A