September: Ovarian

Identifying a new weakness in aggressive ovarian cancer

Joon Kim, MSc and Komila Zakirova, MSc
September 14, 2020

A group of researchers at the London Regional Cancer Program has recently found that ovarian cancer cells perish without a protein called Liver Kinase B1 (LKB1), offering hope for new therapeutic possibilities.

The female reproductive system contains two ovaries located deep in a woman’s pelvis, one on each side of the uterus (womb). The ovaries produce eggs (ova) and female hormones estrogen and progesterone. Among all diseases affecting female reproductive organs, ovarian cancer is the deadliest. In Canada, it is the fifth most common cause of death from cancer in women. Early-stage ovarian cancer, in which the disease is confined to the ovary, rarely causes any symptoms. Due to its asymptomatic nature and lack of reliable detection tests, most patients are diagnosed with a late-stage disease when cancer has already spread (metastasized) beyond the ovaries. At that point, the five-year survival is only 30%, and patients undergo invasive surgery to remove visible cancer clumps and even parts of surrounding organs. They also receive therapy drugs (chemotherapy) that kill cancer cells but have serious side effects. The disease may initially respond to chemotherapy but may return, and if it does, it is often resistant to further chemotherapy. 

Ovarian cancers are named for their cell of origin. Epithelial ovarian cancer (EOC) arises from epithelial cells, which are cells that line the outer surface of organs. It is the most common and most lethal type of ovarian cancer. Patients with late-stage disease frequently develop fluid in the abdominal cavity, and this fluid build-up is known as ascites. EOC tumours shed cancerous (malignant) cells into the ascites where they form multicellular clumps called spheroids. These spheroids attach to and invade nearby organs within the abdominal cavity, further spreading the cancer (Figure 1). Therefore, understanding the mechanisms of spheroid formation (with the goal of stopping this process) is fundamental to improving patient outcomes.  

Figure 1. Epithelial ovarian cancer metastasis(Figure adapted from Peart et al., 2015).

The objective of the work by a group at London Regional Cancer Program was to determine the role of a protein called Liver Kinase B1 (LKB1) in the spread of EOC (Buensuceso et al., 2020). Based on previous work, Buensuceso et al. hypothesized that loss of LKB1 would decrease EOC cell growth and spread. To test this, the protein LKB1 was removed (using the “DNA-scissors” CRISPR-Cas9*) in two groups of human EOC cells (named LKB1-KO), and compared against the original cells, in which LKB1 was not removed

To measure the tumour-forming potential of these cells, the researchers placed them on a specially coated plastic dish to force them to aggregate together and float in suspension as spheroids—mimicking how the primary tumour spreads in patients (Figure 2). A dye that specifically stains only dead cells, not live cells, was used to identify dead cells, subtract them from both dead and live cells to calculate the number of live cells, and the number of live cells was counted under a microscope. In this setting, removing LKB1 significantly decreased the number of surviving cells in the dish for both cell groups compared to the original cells.

Figure 2. Cell culture system that mimics spheroid formation seen in ovarian cancer patient ascites. EOC cells are grown in regular plastic plates on which they can adhere to the surface and rapidly proliferate, which approximates tumour growth at an early stage. These cells are then transferred to specially coated (Ultra-low attachment) plates where they float in suspension. Suspended EOC cells naturally cluster together and form spheroids. Spheroids are subsequently transferred back to standard culture plates to facilitate reattachment, which mimics metastatic spread of the disease. (Figure adapted from Tong et al., 2015).

With this primary evidence that EOC cells cannot survive without LKB1, the hypothesis was tested in a mouse model to see if this idea was true in a living system. LKB1-KO and parental EOC cells were injected into the abdominal cavity of mice and tumour clumps were measured by cutting out and weighing them. In a similar fashion to the first experiment, “LKB1-free” cells formed significantly fewer tumours than the original cells. These experiments provide strong evidence that LKB1 is required for EOC to spread. 

While further research is needed to tease out the exact molecular mechanism that links LKB1 to EOC spread, LKB1’s “pro-tumour” or “tumour-supportive” role in this context is an unexpected finding. LKB1 is found to be frequently missing or rendered inactive by mutations in several cancers such as non-small cell lung cancer, primary lung carcinoma, cervical cancer and skin cancer, which strongly suggests that LKB1 has an “anti-tumour” or tumour-suppressive role. 

This work by Buensuceso et al. has direct implications for the future of EOC therapy. Given that LKB1 has a “pro-tumour” role, the next step is to screen and develop small synthetic molecules that can block or inhibit LKB1’s “pro-tumour” function described by the group. Further research is necessary to find a molecule that specifically targets LKB1 with little to no side effects for the patient. If found, a drug based on the molecule could be used to prevent the spread of EOC from its primary tumour, ultimately improving patient survival.

The researchers who contributed to this work would like to thank the Canadian Institutes for Health Research, London Regional Cancer Program and the London Health Sciences Foundation for various funding. 

*CRISPR-Cas9 is a system that can accurately remove or modify proteins inside a cell. Two pioneers in the field, Drs. Emmanuelle Charpentier and Jennifer Doudna, were jointly awarded the Nobel Prize in Chemistry for their discovery in 2020. 

Original article:

Buensuceso, A., Ramos-Valdes, Y., DiMattia, G. E., & Shepherd, T. G. (2020). AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis. Molecular Cancer Research, 18(3), 488–500. https://doi.org/10.1158/1541-7786.MCR-19-0530

References

Brett M., R., Jennifer B., P., Thomas A., S., Brett M., R., Jennifer B., P., & Thomas A., S. (2017). Epidemiology of ovarian cancer: A review. Cancer Biology & Medicine, 14(1), 9–32. https://doi.org/10.20892/j.issn.2095-3941.2016.0084 http://www.cancerbiomed.org/index.php/cocr/article/view/1004/1122

Peart, T., Valdes, Y. R., Correa, R. J. M., Fazio, E., Bertrand, M., McGee, J., Préfontaine, M., Sugimoto, A., DiMattia, G. E., & Shepherd, T. G. (2015). Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids. Oncotarget, 6(26), 22424–22438. https://doi.org/10.18632/oncotarget.4211

Reid, B. M., Permuth, J. B., & Sellers, T. A. (2017). Epidemiology of ovarian cancer: A review. 14(1), 24. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365187/Tong, J. G., Valdes, Y. R., Barrett, J. W., Bell, J. C., Stojdl, D., McFadden, G., McCart, J. A., DiMattia, G. E., & Shepherd, T. G. (2015). Evidence for differential viral oncolytic efficacy in an in vitro model of epithelial ovarian cancer metastasis. Molecular Therapy – Oncolytics, 2, 15013. https://doi.org/10.1038/mto.2015.13