The role of Wnt5a in breast cancer cell metabolism

Abstract

Introduction
Breast cancer metastasis has been coupled to deregulated cell signaling pathways, among them the non-canonical Wnt pathway. Wnt5a is a non-canonical Wnt ligand which expression is downregulated in many metastatic breast tumors. It is thought to function as a tumor suppressor of breast cancer metastasis by regulating breast cancer cell migration through, for example, increased adhesion. It has been shown that Wnt5a increases aerobic glycolysis in melanoma cells, where it acts as a tumor promoter. However, Wnt5a is suggested to increase oxygen consumption rate in breast cancer cells. We therefore investigated effects of Wnt5a on aerobic glycolysis and oxidative phosphorylation in breast cancer cells, and Wnt5a-mediated reduction of breast cancer cell migration.

Methods
The model system employed includes both parental MCF7 cells negative for Wnt5a protein expression and the transfected breast cancer cell line MCF7-5A, which constitutively expresses the Wnt5a protein. The MCF7-5A cells were analyzed for migration using wound healing assay, and for c-myc, hexokinase II, LDHA and mitochondrial protein (complex I-V) expression using Western blot analysis, and for lactate secretion using a colorimetric lactate assay. The same experimental procedures were conducted for EGF treated MCF7-5A cells.

Results
Wnt5a reduced MCF7-5A cell migration, compared to MCF7-EV control cells. The expression levels of c-myc, hexokinase II and LDHA, lactate secretion and the expression of mitochondrial protein complex I-V were unchanged in MCF7-5A cells in the presence of Wnt5a, compared to control. MCF7-5A cells showed increased wound healing ability when treated with EGF for 48 hours, as compared to MCF7-EV cells. Furthermore, the β-catenin and c-myc expression levels increased slightly in MCF7-5A cells upon EGF treatment. No difference in levels of hexokinase II, LDHA, lactate, or mitochondrial complex I-V protein was observed in EGF treated MCF7-5A and MCF7-EV cells.

Conclusions
Our results suggest that Wnt5a mediated reduction in MCF7 cell migration is not dependent on aerobic glycolysis. It is still possible that Wnt5a mediates effects on the oxidative phosphorylation but it is most likely not through regulating mitochondrial complex I-V protein expression.

Popular Abstract

Wnt5a and its role in breast cancer progression

When cancer spreads and localizes to other distant organs apart from its primary origin it is known as cancer metastasis. The underlying cause that allows tumor cells to spread is most likely a combination of both gaining and loosing fundamental mechanisms that regulate the cancer cell behavior.

Wnt5a is a signaling molecule that is involved in controlling the movement of cells during early embryonic development. It is also involved in the movement of tumor cells and its expression is often found to be missing in metastatic breast cancer. It appears that Wnt5a can act as a “stop” signal to inhibit breast cancer cells to move and give rise to new tumors, possibly through making the breast cancer cells attach more tightly to each other.

Cancers consume more energy compared to normal and healthy tissues. This abnormal behavior could be another cause for tumor cell metastasis. In our project, we investigated if Wnt5a can control the movement of breast cancer cells by controlling their energy metabolism. Lactate is a metabolic end product of a process known as aerobic glycolysis, where glucose is stepwise broken down to lactate in the presence of oxygen. Lactate is often plentiful in tumors and is a probable contributor to tumor metastasis. To observe whether Wnt5a control aerobic glycolysis in breast cancer cells, we looked at lactate secretion in the MCF7 breast cancer cell line, both in the presence and absence of Wnt5a. The MCF7 cell line was established in the 1970s and comes from a woman with metastatic breast cancer. We also studied if Wnt5a had any effects on the levels of different proteins involved in the aerobic glycolysis (proteins such as c-myc, hexokinase II and LDHA). We additionally investigated if Wnt5a could shift the energy metabolism away from glycolysis to oxidative phosphorylation (OXPHOS), which is the major pathway for generating the cellular energy currency ATP.

Our results show that Wnt5a indeed control the movement of MCF7 cells. However, we observed that the lactate production remained the same in the presence of Wnt5a, as compared to control (i.e. in absence of Wnt5a). We could not observe a difference in the protein concentration of glycolytic proteins (c-myc, hexokinase II and LDHA), in the presence of Wnt5a. We therefore believe that Wnt5a control the movement of MCF7 breast cancer cells by other means than controlling the lactate production. The MCF7 cell line naturally produce less lactate than other breast cancer cell lines. We therefore increased the lactate production in MCF7 cells with a substance known as epidermal growth factor (EGF). In this setting, we could not notice an effect of Wnt5a on aerobic glycolysis. The additional experiments using EGF support our belief that Wnt5a regulate MCF7 cell movement through mechanisms other than alteration of aerobic glycolysis. We also observed that the concentration of the proteins involved in OXPHOS were unaffected by Wnt5a. It suggests that Wnt5a control the MCF7 cell movement independent of changing the levels of the OXPHOS proteins.


Advisor: Tommy Andersson
Master´s Degree Project 30 credits in Medical Biology 2014
Department of Biology, Lund University

• author: Södergren, Katja
• supervisor: Tommy Andersson
• organization: Degree Projects in Molecular Biology
• course: MOBM01 20142
• year: 2015
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 5050967
• date added to LUP: 2015-02-17 16:12:45
• date last changed: 2015-02-17 16:12:45