WNT5A signaling and MARCKS phosphorylation in melanoma cell migration and invasion
(2019) MOBM02 20182Degree Projects in Molecular Biology
- Abstract
- The positive correlation between elevated expression of wingless-type mammary tumor virus integration site family member 5A (WNT5A) and metastatic potential in melanoma has been extensively explored. However, detailed mechanistic aspects regarding how WNT5A promotes increased melanoma aggressiveness remains unknown. Myristoylated alanine-rich C-kinase substrate (MARCKS), an intracellular protein capable of reversible actin cross-linking in a phosphorylation-dependent manner, has been implicated in the progression of various cancers, including melanoma. Here, we demonstrate the potential relationship between WNT5A signaling and MARCKS phosphorylation in melanoma, focusing on cell motility and invasiveness. First, WNT5A and MARCKS... (More)
- The positive correlation between elevated expression of wingless-type mammary tumor virus integration site family member 5A (WNT5A) and metastatic potential in melanoma has been extensively explored. However, detailed mechanistic aspects regarding how WNT5A promotes increased melanoma aggressiveness remains unknown. Myristoylated alanine-rich C-kinase substrate (MARCKS), an intracellular protein capable of reversible actin cross-linking in a phosphorylation-dependent manner, has been implicated in the progression of various cancers, including melanoma. Here, we demonstrate the potential relationship between WNT5A signaling and MARCKS phosphorylation in melanoma, focusing on cell motility and invasiveness. First, WNT5A and MARCKS transcription levels were found to be higher in phenotypically invasive melanoma cells. Further investigation of four human melanoma cell lines showed an increased endogenous protein expression of both unmodified and phosphorylated MARCKS in invasive WM852 cells, known to produce copious amounts of WNT5A. A more direct connection between WNT5A and MARCKS was revealed after treating A375 and A2058 cell lines with recombinant WNT5A, with increased MARCKS phosphorylation being evident after 15 and 30 minutes. Moreover, siRNA-based WNT5A silencing in A375 cells caused decreased MARCKS phosphorylation. WNT5A-induced MARCKS phosphorylation was efficiently restrained in A2058 cells upon inhibiting protein kinase C (PKC) and Rho-associated protein kinase (ROCK). Notably, ROCK inhibition led to a more prominent decrease in MARCKS phosphorylation, with no observed additive effects when hindering the activity of both kinases. Our conclusive experiments demonstrated that the stimulatory effects of WNT5A regarding cell migration and invasion were limited after siRNA knockdown of MARCKS in A375 cells, suggesting that MARCKS might be involved in triggering metastatic behavior. Altogether, our data indicate that WNT5A signaling, via PKC and ROCK, induces MARCKS phosphorylation in melanoma cells, a process appearing to be significant for migration and invasion. Thus, though necessitating additional research, targeting MARCKS could be of therapeutic importance in the treatment of metastatic melanoma. (Less)
- Popular Abstract
- WNT5A and MARCKS – partners in the crime of melanoma cancer?
Like a reckless monster, cancer may spontaneously attack anyone, anywhere, at any time, making the battle against it a daunting task. So, what should we do to ensure a win? Well, knowledge is power. Finding out how your opponent operates is vital in order to build a good combat strategy, preferably attacking at the most critical points.
Acting as a protective barrier against the outer world, our skin, just like every other organ, may be struck by cancer. Out of the numerous different types of skin cancer, few are as deadly as melanoma, able to spread through the body like wildfire if not contained at early stages. In other words, we desperately need to learn more about the... (More) - WNT5A and MARCKS – partners in the crime of melanoma cancer?
Like a reckless monster, cancer may spontaneously attack anyone, anywhere, at any time, making the battle against it a daunting task. So, what should we do to ensure a win? Well, knowledge is power. Finding out how your opponent operates is vital in order to build a good combat strategy, preferably attacking at the most critical points.
Acting as a protective barrier against the outer world, our skin, just like every other organ, may be struck by cancer. Out of the numerous different types of skin cancer, few are as deadly as melanoma, able to spread through the body like wildfire if not contained at early stages. In other words, we desperately need to learn more about the disease, allowing us to put out the fire out before it is too late. An already well-known contributor to the aggressiveness of melanoma cells is WNT5A, a protein affecting the inner workings of cells from the outside. However, while we know that it definitively causes trouble, we are not exactly sure how or, importantly, with the help of who. Therefore, in an effort to make the fight against melanoma more efficient, we have tried to identify and explore a possible companion of WNT5A in melanoma – a protein called MARCKS.
Owing to its ability to modulate the cellular skeleton, MARCKS regulates processes such as adhesion, motility, and secretion – all important in the making of an aggressive and invasive cancer cell. To do this, MARCKS is dependent on a specific activation signal, changing and moving the protein inside the cell. Now, might WNT5A, like an outside traffic controller, trigger this signal in melanoma cells? To investigate this possibility, we started by searching in a large online database, revealing that the mRNA levels of WNT5A and MARCKS are high in invasive melanoma cells. With mRNA being the direct blueprint for creating proteins, this indicated to us that both proteins could be important for invasion. Moving on to studying the actual proteins, we found large amounts of MARCKS, with and without the activation signal, in a particularly invasive melanoma cell line, known to produce a lot of WNT5A. What is more, supplying melanoma cells with external WNT5A gave a considerable increase in MARCKS activation.
In short, our first series of experiments provided solid support for the notion that WNT5A activates MARCKS in melanoma cells. Additionally, we also transiently removed WNT5A from our cells, resulting in decreased activation of MARCKS, and we were able to manipulate MARCKS activation by inhibiting known associates of WNT5A. Simply put, all of these results point towards WNT5A and MARCKS having a relationship. But, one might wonder, is this relationship relevant in causing melanoma cell aggressiveness? According to our final assays, the answer is yes! When we challenged MARCKS-depleted cells to migrate and invade, external WNT5A was not able to increase cellular invasiveness, suggesting that MARCKS is necessary for WNT5A to cause trouble. Altogether, the present study provides deeper insight regarding the dangers of WNT5A in melanoma, identifying a potentially vital partner in crime. With future research, MARCKS could become one of those critical points of attack, enabling us to battle against and stop invading melanoma cells.
Master’s Degree Project in Medical Biology, 30 credits, 2019
Department of Biology, Lund University
Supervisors: Tommy Andersson and Purusottam Mohapatra
Division of Cell and Experimental Pathology, Department of Translational Medicine, Lund University, Clinical Research Centre, Skåne University Hospital, SE-20213 Malmö, Sweden (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8974287
- author
- Toftdahl, Maren
- supervisor
- organization
- course
- MOBM02 20182
- year
- 2019
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 8974287
- date added to LUP
- 2019-04-09 10:58:31
- date last changed
- 2019-04-09 10:58:31
@misc{8974287,
abstract = {{The positive correlation between elevated expression of wingless-type mammary tumor virus integration site family member 5A (WNT5A) and metastatic potential in melanoma has been extensively explored. However, detailed mechanistic aspects regarding how WNT5A promotes increased melanoma aggressiveness remains unknown. Myristoylated alanine-rich C-kinase substrate (MARCKS), an intracellular protein capable of reversible actin cross-linking in a phosphorylation-dependent manner, has been implicated in the progression of various cancers, including melanoma. Here, we demonstrate the potential relationship between WNT5A signaling and MARCKS phosphorylation in melanoma, focusing on cell motility and invasiveness. First, WNT5A and MARCKS transcription levels were found to be higher in phenotypically invasive melanoma cells. Further investigation of four human melanoma cell lines showed an increased endogenous protein expression of both unmodified and phosphorylated MARCKS in invasive WM852 cells, known to produce copious amounts of WNT5A. A more direct connection between WNT5A and MARCKS was revealed after treating A375 and A2058 cell lines with recombinant WNT5A, with increased MARCKS phosphorylation being evident after 15 and 30 minutes. Moreover, siRNA-based WNT5A silencing in A375 cells caused decreased MARCKS phosphorylation. WNT5A-induced MARCKS phosphorylation was efficiently restrained in A2058 cells upon inhibiting protein kinase C (PKC) and Rho-associated protein kinase (ROCK). Notably, ROCK inhibition led to a more prominent decrease in MARCKS phosphorylation, with no observed additive effects when hindering the activity of both kinases. Our conclusive experiments demonstrated that the stimulatory effects of WNT5A regarding cell migration and invasion were limited after siRNA knockdown of MARCKS in A375 cells, suggesting that MARCKS might be involved in triggering metastatic behavior. Altogether, our data indicate that WNT5A signaling, via PKC and ROCK, induces MARCKS phosphorylation in melanoma cells, a process appearing to be significant for migration and invasion. Thus, though necessitating additional research, targeting MARCKS could be of therapeutic importance in the treatment of metastatic melanoma.}},
author = {{Toftdahl, Maren}},
language = {{eng}},
note = {{Student Paper}},
title = {{WNT5A signaling and MARCKS phosphorylation in melanoma cell migration and invasion}},
year = {{2019}},
}