Mapping the expression of the Tribble family of pseudokinases in neuroblastoma

Abstract

Neuroblastoma (NB) is a highly heterogeneous childhood cancer where the presence of chemotherapy resistant cancer stem cells (CSCs) contributes to a high relapse rate in high-risk patients. Understanding the mechanisms by which CSCs contribute to therapy resistance could lead to future treatment strategies. The human pseudokinase TRIB3 has been shown to support stemness and play a role in cell cycle arrest. In this study, we investigated the expression of the Tribble family with a special focus on TRIB3, and its possible correlation to cancer stemness and proliferation in NB. Low TRIB3 mRNA expression was observed in all tested NB cell lines, whereas a higher fraction of cells, in all cell lines, expressed the TRIB3 protein. Our results suggested that the TRIB3 expression correlates to proliferating cells (Ki67+) and to stem cell marker Nestin in the two high-risk resistant NB cell lines BE(2)-C and SK-N-FI. Interestingly, BE(2)-C is a cell line that has been suggested as a possible NB CSC population. Furthermore, we used a novel Multiplex imaging technology for staining of multiple markers in NB organoids representing a more accurate model of tumour growth. TRIB3 was not detected in this material, and final conclusions regarding the Multiplex imaging results require further protocol optimization of the technology. In conclusion, the data supports a co-expression between TRIB3, stemness, and proliferation in 2D cultures, with further studies needed to confirm the role of TRIB3 in 3D cultures. Investigating TRIB3 and its role in NB stemness and cell cycle could aid high-risk patients and possibly reduce relapse rates.

Popular Abstract

Neuroblastoma (NB) is a childhood cancer where the presence of chemotherapy resistant cancer stem cells contributes to a high relapse rate in high-risk patients. This study investigated the human Tribbles homolog TRIB3 and its possible correlation to NB stemness and proliferation.

NB is a tumour of the sympathetic nervous system and it accounts for approximately 10% of all pediatric cancers. The majority of children diagnosed with NB have a high survival probability. However, a group of patients diagnosed with high-risk NB have a higher risk of relapse and survival after relapse is currently <10% over a five year period. A major difficulty with cancer treatment is therapy resistance and one of the underlying mechanisms for this might be the presence of a small fraction of cancer stem cells. These cancer stem cells are considered a subset of cells with the ability to self-renew and differentiate into new cancer cells, driving growth and progression of the cancer. Two markers associated with cancer stem cells are SOX2 and Nestin, and an expression of both these markers has been found in NB.

Pseudokinases are inactive enzymes, lacking the typical functions of the kinase enzymes. The human Tribble family consists of three Tribble pseudokinases and these are involved in various signaling pathways and are often found dysregulated in disease. Interestingly, studies have shown that TRIB3 supports stemness (a word used to describe stem cell-properties) in breast cancer and also plays a role in cell cycle arrest.

These findings raise the question if TRIB3, one of the Tribbles, can play a role in supporting stemness and proliferation in NB. We studied the expression of the Tribble family of pseudokinases in NB cell lines with a special focus on TRIB3 expression and its correlation to stemness markers SOX2 and Nestin as well as a marker for proliferation. Online databases confirmed a positive correlation between high TRIB3 expression and a lower survival probability. The expression of the different markers was studied at both gene and protein level, and overall low levels of TRIB3, SOX2, and Nestin were found. Despite this, we found that the number of cells double positive for TRIB3 and Nestin were highest in two cell lines, one of which has been identified as having stemness-like properties and the other which has been determined as a cell line highly resistant to the chemotherapy drug doxorubicin. This suggests a possible correlation between upregulation of TRIB3 in cells expressing stemness-like properties and cell lines showing a lower sensitivity towards chemotherapy. Additionally, almost all TRIB3+ cells were also positive for the proliferation marker, indicating that TRIB3 is expressed in only dividing cells.

Furthermore, we cultured 3D organoids, which are “organ-like” clumps of cells, to create a more accurate model of the tumor growth in NB. Small organoids were cultured, and although the protocol for the organoid-culturing needs further optimization, this is a promising start of studying cell interactions in a 3D structure using cell lines as a base to start with. We also used a novel imaging technology allowing for investigation of multiple markers to obtain a more complex visualization of NB cells grown as organoids.

This study gives an exciting introduction to TRIB3, and more specifically, how it can play a role in NB stemness and proliferation. Future research on TRIB3 and its mechanisms might contribute to understanding the mechanisms behind high-risk NB and thus lead to new treatment strategies.

• author: Halipi, Vesa
• supervisor: Kathrin Zeller
• organization: Department of Immunotechnology
• course: KIMM05 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• language: English
• additional info: Jag har skickat med blanketten som ska skrivas under av institutionen
• id: 9059336
• date added to LUP: 2021-06-30 08:28:26
• date last changed: 2021-06-30 08:28:26