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In vivo selection reveals autophagy promotes adaptation of metastatic ovarian cancer cells to abdominal microenvironment

Ovarian cancer is the second most common cause of gynaecologic cancer death in women around the world. The mortality‐to‐incidence ratio in ovarian cancer is >0.6 and studies from US and UK registries estimate that 1 in 6 women die within the first 90 days of diagnosis. The disease is often diagnosed late and composed of several subtypes with distinct biological and molecular properties, even within the same histological subtype, with limited treatment options. Although early‐stage disease is highly curable the majority of women present with metastatic, chemo-resistant disease due in part to the lack of effective screening options to detect ovarian cancer at an early stage and a lack of early, specific warning signs or symptoms.

The precise molecular mechanisms that promote ovarian cancer dissemination remain undefined. Nevertheless, it is becoming clear that it can disseminate passively by movement of peritoneal fluid in the abdominal cavity. In addition, ovarian cancer is often accompanied by malignant ascites, which can be found in more than one-third of patients at the time of diagnosis and in almost all patients at recurrence. The forming ascites further provides a complicated liquid environment that not only accelerates transportation of cancer cells to distant sites but also supports their survival before implantation.

Autophagy is an intracellular degradative process that occurs under several stressful conditions including organelle damage, the presence of abnormal proteins, and nutrient deprivation. The mechanism of autophagy initiates the formation of autophagosomes that capture degraded components and then fuse with lysosomes to recycle these components. The modulation of autophagy plays dual roles in tumour suppression and promotion in many cancers. In addition, autophagy regulates the properties of cancer stem-cells by contributing to the maintenance of stemness, the induction of recurrence, and the development of resistance to anticancer reagents.

It has been widely accepted that tumour masses contain heterogeneous subpopulations of cancer cells and these subpopulations have different potentials in terms of malignancy. Thus, it can be postulated that the outcome of ovarian cancer metastasis may result from the survival and expansion of specialised colonies with particular properties that pre-exist in the parental tumour and have then undergone multiple selective steps. The molecular mechanism behind this adaptation however, is unclear and is explored in a recent study by Kuo et al.

The authors established an in vivo mouse peritoneal dissemination scheme that allowed them to select more aggressive sublines from the parental ovarian cancer cells A2780 and SKOV‐3. After the first intraperitoneal implantation of the cell lines into nude mice peritoneal metastatic nodules were harvested at specific time-points after xenografting, processed and implanted into new hosts. This procedure was repeated three times and yielded the sublines called A2780-M3 and SKOV-3-M3, respectively. The tumourgenicity and growth characteristics of these sublines were investigated and microarray and differential gene expression analyses were performed.

The sublines A2780-M3 and SKOV-3-M3 were more aggressive than their corresponding parental cell line in vivo, where the tumour burden and the number of metastatic nodules were significantly increased after intra peritoneal engraftment in nude mice.

An in vitro anchorage-independent growth assay showed that SKOV-3-M3 exhibited a higher ability to undergo anchorage-independent growth compared to the parental cell line.

The A2780-M3 cells showed morphological growth changes in vitro and formed clusters with a round morphological shape rather than adhering to the tissue culture dish like the parental cells.

Furthermore, the A2780-M3 subline grew significantly faster than the corresponding parental line under high stress, caused by culturing the cells at high density (5x106 cells/cm2), and showed decreased mRNA expression of the pro-apoptotic markers BAD and APAF1 and increased expression of the anti-apoptotic marker BCL2L1.

cDNA microarray (Affymetrix GeneChip Human Genome U133 Plus 2.0 Array) was performed to determine differentially expressed genes between A2780-M3 and its parental A2780 and gene set enrichment analysis (GESA) revealed that autophagy related genes are enriched in A2780-M3 (GO_AUTOPHAGOSOME, normalised enrichment score (NES) =1.87: FDR=0.13).

A2780-M3 cells showed a greater accumulation of LC3-II and exhibited more autophagic puncta than A2780 under stress-induction by serum starvation or after exposure to cisplatin.

Furthermore, the autophagic inhibitor wortmannin, significantly reduced the growth of A2780-M3 cells but not the parental A2780 cells under high-cell density conditions.

In a clinical cohort of 1656 ovarian cancer patients Kaplan-Meier analysis demonstrated that 73% (22/30) of the autophagy related genes identified correlated with patient survival rates.

The findings in this paper indicate that an increase in autophagic potency among ovarian cancer cells is crucial for selection of metastatic colonies in the abdominal microenvironment. The ovarian cancer sublines established by the Authors (A2780-M3 and SKOV-3-M3), showed a higher tumourgenicity and the ability to undergo autophagic induction under a variety of stress conditions leading to increased proliferation and survival. Taken together, autophagy may be a promising potential therapeutic target in the treatment of ovarian cancer.

Reference

Kuo et al., 2019. In vivo selection reveals autophagy promotes adaptation of metastatic ovarian cancer cells to abdominal microenvironment. Cancer Science 00:1-11

https://doi.org/10.1111/cas.14162

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