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by Jacqui

Can we edit GABRA3 to suppress AKT activation and therefore metastasis in breast cancer?

March 11, 2016 in Uncategorized

Around 58,000 women and men are diagnosed with breast cancer each year in the UK and out of this 11,500 dying from this disease. With the rate of breast cancer increasing by 3.5% a year it’s never been more important to find a way of reducing these statistics.

Why is Gabra3 important?

This study on Gabra3 suppression looks into identifying the processes in which metastasis occurs and ways to reduce it.

GABAa is an inhibitory neurotransmitter in the brain that acts as a channel in response chemicals binding. This study focused on GABAa receptor alpha 3 or Gabara3 normally found in the brain however has been found in metastatic breast cancer. This receptor is thought to kick start the AKT pathway.  It’s this pathway that stimulates cell migration and therefore metastasis.

Metastasis is a secondary tumour away from the initial site, and is the cause of most breast cancer fatalities. Which is why finding a way to reduce metastasis is important as this will reduce the ever increasing death toll.

Studying GABRA3 is vital as it’s only found in metastatic cancer tissues and as it’s a cell surface molecule has the potential to be a biochemical target. Currently being used to treat hypersomnia and therefore has the FDA approval making it more economically viable.

Experimenting with Gabra3

Cells expressing Gabra3 found in the MCF-7 cell line showed an increase in cell migration. To measure this MCF-7 cells were tagged with a biochemical marker and inserted into mice mammary fat pads.

mice 2

Shows the primary tumour and lung metastasis in mice shown using a Xenogen bioluminescence technique.

Mice that had the MCF-7 GABRA3 gene all developed lung metastasis as a result whereas the control mice showed no metastasis. This confirms that Gabra3 is having a significant effect and results in metastasis.

By altering the MDA-MB-436 breast cancer cells that express Gabra3 with the addition shRNA, which is a short RNS sequence that has a kink in it. This is used to silence target genes, this edited MDA-MB-436 Gabra3 shRNA should suppress Gabra3 and top the AKT pathway.

This edited gene was tested using mice again as the MDA-MB-436 that expressed the edited MDA-MB-436 shRNA and a control shRNA was inserted into the mammary fat pads.

mice 2

Shows the lung metastasis in the edited MDA-MB-436 shRNA and the control, shown using a Xenogen bioluminescence technique.

The results of this experiment show that 90% of the mice with the control shRNA developed lung metastasis. Whereas the mice with the shRNA edition only 29% developed lung metastasis. This shows that the edited MDA-MB-436 cells reduces the expression of Gabra3 reducing metastasis and increases survivability. Exactly how the shRNA edition suppresses the effects of Gabra3 is unknown and requires further experimentation.

But the body created has its own edition of Gabra3 that stops the AKT pathway activation. At the I/M site in the brain the Gabra3 undergoes A-to-I RNA Gabar3 edition. Unfortunately this edition only occurs in non-invasive (or primary) breast cancers and not in the metastatic cancer, however understanding how this edition occurs could be used to produce a potential drug.

 

By examining the differences between the non-invasive and metastatic breast cancer cells it was found that two specific enzymes operate this edition. These are ADAR1 and ADAR2 but its ADAR1 that is most significant.

ADAR1 generates the ADAR1p110 enzyme, this enzyme was found in the breast cancer cells along with the normal human epithelial cell line (HMEL). Whereas the other ADAR1 enzyme ADAR1p150 and ADAR2 were not found in the cell lines. This indicates that it’s the ADAR1p110 enzyme that causes the A-to-I Gabra3 edition.

To understand the effect of the A-to-I Gabra3 edition RNA edited Gabra3 with an A-to-G mutation was inserted to the I/M site in MDA-MB-436 cells in vitro expressing the unedited Gabra3. Cell migration and therefore metastasis was considerably reduced.

Leading from this the A-to-I edited Gabra3 was tested in vivo to see if it had similar affects as the in vitro experiment. Tagged MDA-MB-436 cells expressing the A-to-I edition plus a control were injected into mice.

Metastasis in mice 3

A-to-I edited Gabra3 and control injected into mice, shown using a Xenogen bioluminescence technique.

From this you can see that the control shows a high aggregation on metastatic cells, compared to the edited Gabra3 which shows a considerable difference in metastasis proving that the A-to-I edited Gabra3 has a positive effect in reducing metastasis.

The edited edition of Gabra3 works because the unedited Gabra3 is a cell surface molecule and the A-to-I edited Gabra3 decreases the expression of the Gabra3 therefore reduces the activation of the AKT pathway so cell migration reduced.

Edited and unedited Gabra3 were in vivo tested in MDA-MB-436 cell line to look at the interaction between the two Gabra3 molecules. They were added in an equal 1:1 ratio, the results of this show that the edited Gabra3 has the ability to suppress AKT pathway activation as a result of the unedited Gabra3 by 50%.

How Gabra3 causes metastasis is unknown, it’s been proven to activate the AKT pathway causing series of chain reactions that leads to cell migration. Stem cells are believed to contribute extensively to metastasis, Gabra3 increases this stem cell production. By observing the stem cell population with the addition of the edited Gabra3 it showed that the edited Gabra3 actually has no effect on stem cell populations. This also confirms that it is the unedited Gabra3 causing a substantial increase in stem cell population.

Future importance

Patient fatality due to breast cancer is either therapy resistance or the development of metastasis. That’s why this study is so important with options to either target Gabra3 specifically using shRNA or increasing the A-to-I edited Gabra3 production.

As Gabra3 is already a tried and tested molecule it already has FDA approval increasing its viability. In addition with this new evidence showing that Gabra3 has a significant importance and effect on cell migration and metastatic development further testing and experimentation on Gabra3 holds the solution to reducing patient fatalities caused by breast cancer.

 

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by Tanya

The lasting effects of PCBs on two isolated populations of Atlantic and Pacific Orca.

March 11, 2016 in Uncategorized

The Orca

The orca (Orcinus Orca) is easily the most recognisable of all cetaceans (whales, dolphins and porpoises) in the world. Its striking black and white colouration has captured the hearts of many, both young and old, over recent decades.

The now rare sight of an orca calf, unlikely to be seen in Eurpoean waters again.

Figure 1 – The now rare sight of an orca calf, unlikely to be seen in European waters again.

The orca is widely distributed throughout the world’s oceans. In the Pacific Ocean, the orca is an important natural symbol for the people of British Colombia. It is here that two harmonious populations with differing diets live. These populations consist of the transient orcas that are mostly marine mammal eating and the resident population which feed on fish. Over in the Atlantic Ocean the orca is less numerous and only a small resident population is found in industrialized regions of Europe around the Mediterranean Sea. This resident population is primarily tuna-feeding. Also, a small group of marine mammal eating orca is regularly seen around western Ireland and North West Scotland. These four populations of orca are all heavily monitored for research purposes.

However, in a recent paper in Nature Communications it has been proposed that these populations are under threat from an almost silent enemy, PCB pollution.

What is PCB?

Polychlorinated biphenyls (PCBs) are a major toxic industrial chemical that was produced during the twentieth century. PCB is well known for its environmental toxicity and its classification as a persistent organic pollutant. It was in the 1960s that PCBs where first identified as a problem. They were then subsequently banned in America in 1979 and in Europe in 1981. More recently they were banned by the Stockholm Convention on Persistent Organic Pollutants in 2001.

PCB is known to have numerous effects on humans including being carcinogenic. Not only can PCBs and other persistent toxins affect humans but they are having increasing and lasting effects on other organisms, especially those that are top predators in marine food chains.

Marine mammals that inhabit coastal waters of Europe, the Mediterranean Sea and the waters around Vancouver Island have been found to be highly contaminated with not only PCBs but also other toxic pollutants like dichlorodiphenyltrichloroethane (DDT). These chemicals accumulate in the fatty tissues of these animals.

The evidence is increasingly supportive that the increased concentration of PCBs in the ocean is leading to an increase in abnormalities seen in numerous marine species. Such abnormalities include: skeletal deformities, hormone disruption, immunotoxicity, and reproductive failure.

One majestic marine organism that is feeling the increasing effects of PCB in the world’s oceans in very different ways is the orca.

Effects of PCBs

Both the Pacific and Atlantic populations of orca are being affected by PCBs. It has been found that the PCB concentrations in individual male Pacific resident orca increases with age. This is due to them gaining PCB from their prey, which are also contaminated with this toxin. Contrastingly, the PCB concentrations of females in resident populations is greatly reduced with reproductive activity. The females pass on PCB to their offspring when in the womb and during lactation thus, reducing their own levels of PCB contamination. This is evident in many populations of orca where females are reproducing normally, and the concentrations of PCB is seen to decrease significantly with each successful pregnancy. The transient Pacific orca are particularly contaminated with PCBs due to their dietary preference of marine mammals, which themselves are high in PCB contamination. These two Pacific populations are considered to be among the most contaminated cetaceans in the world.

The two monitored populations of orca in European waters are split into almost identical categories as the Pacific orca, the Scottish population been marine mammal eating like the transients, and the southern population been predominantly fish-eating. This allows for good comparison between the Pacific and Atlantic populations.

Over the last 19 years that the Scottish population has been studied there has not been a single report of a calf seen within the group. Similarly for the resident Atlantic population between 1998-2011 only 5 calves were successfully produced and survived beyond a year. This means that over a 13 year period the southern population had a reproductive success rate of only 6.4%. This is one of the lowest recorded reproductive rates for orcas across the globe.

Adult females in the Atlantic consistently have substantially great concentrations of PCBs in their bodies than any individual Pacific orca. The adult males in the Atlantic also have higher PCB concentrations than the resident Pacific orcas and only slightly lower concentrations than the Pacific transients. The high concentrations of PCB in the adult females in the Atlantic is consistent with the reproductive failure seen in the Atlantic populations.

It is evident that the Atlantic females are either having trouble offloading PCBs to their calves during pregnancy or that they may be reaccumulating PCBs in their diets.

What now?

It is clearly evident that whilst the use of PCBs has been banned since the early 1980s, orcas are still continuing to being found heavily contaminated with this deadly toxin. Over the years the concentrations found in orca has decreased. Nevertheless this is a painstakingly slow process. The current levels of PCB in the Pacific orca has allowed them to be classified as among the most contaminated cetaceans in the world.

It is obvious that PCB levels represent an increasing toxicological risk to the Atlantic orcas. They are more heavily affected through the lack of reproduction which is having an increasingly dramatic effect on their population numbers and the future survival of these populations. It is nearing the point where if action is not taken to ascertain why PCB levels are not falling in the Atlantic Ocean that they will become under threat of extinction due to reproductive failure.

The levels of PCB in the Pacific has been declining over recent decades, however this trend has not been duplicated in the Atlantic, we must look into why this is or we may lose the Atlantic orca forever.