Lipids on the Mind

Blog Spotlight #1

Access the article: https://www.nature.com/articles/s41598-018-26636-6.pdf?origin=ppub


Image obtained from: Lacombe, R. J. Scott, Raphaël Chouinard-Watkins, and Richard P. Bazinet. “Brain Docosahexaenoic Acid Uptake and Metabolism.” Molecular Aspects of Medicine, Dietary fatty acids, lipid mediators, cell function and human health, 64 (December 1, 2018): 109–34. https://doi.org/10.1016/j.mam.2017.12.004.

               In the United States alone, more than 200,000 people are diagnosed with brain cancer every year, a common metastasis site for patients with advanced primary lung cancer, breast cancer, or melanoma. Many of these metastases result from other primary sources and approximately 50% of lung and melanoma patients and 20% of breast cancer patients develop secondary lesions in the brain. A common feature in most brain metastases is resistance to therapy, which can be attributed to the poor penetration of therapeutics across the blood-brain barrier (BBB). This research is most impactful since there is very little understood about the mechanisms that regulate BBB permeability in normal brain tissue or brain malignancies. This prevents exploitation of the BBB for drug delivery, a method that could be useful for the treatment of many neurodegenerative or malignant brain diseases. Animal models make tumor pathophysiology comparison difficult, relying on both mouse and human cell lines for research purposes.

               Major facilitator superfamily domain‐containing protein‐2a (Mfsd2a) has recently gained the spotlight for its regulatory role in the maintenance of proper functioning of the BBB (Ocak et al., n.d.). Remarkably, Mfsd2a has been implicated in the mediation of the blood–brain barrier (BBB) permeability by selective transportation of the lysophosphatidylcholine (LPC) fatty acids (Nguyen, et al., 2014). The gene, MFSD2a plays an important role in mammalian tissue and organ growth, lipid metabolism and cognitive and motor functions. Specifically, in the brain and retina, Mfsd2a selectively transports the omega-3 fatty acid docosahexaenoic acid (DHA) across the BBB. This was confirmed by experimenting with a genetic deletion of Mfsd2a mice which led to impaired DHA transport and reduced levels of certain lipid metabolites. Loss-of-function studies provide concrete evidence in support of pursuing this pathway.

               Loss-of-function (including familial mutations) in human MFSD2A are linked to cognitive deficits and ataxia due to deficiencies in DHA transport and metabolism. As we learned in BCM441, DHA cannot be synthesized in the brain, thus it must be transported there by other means. DHA accumulated in the brain is selectively derived from the plasma where it is bound to albumin as an unesterified fatty acid or to LPC. DHA is a crucial polyunsaturated omega‐3 fatty acid required for brain development, motor, and cognitive functioning. A previous study reported down-regulation of Mfsd2a in a pericyte-deficient mouse model, suggesting that pericytes are necessary for the induction or maintaining Mfsd2a gene expression. For this reason, the authors set out to investigate the association between pericytes and endothelial cells within the vasculature of metastatic brain tumors.

Figure 1. The regulation of BBB permeability is mediated by Mfsd2a suppression of caveolae‐mediated transcytosis in endothelial cells. Essential DHA is absorbed from the intestines or synthesized, conjugated to LPC in the liver and bound to albumin forming LPC‐DHA in the plasma. The significant pathway of LPC‐DHA uptake across the endothelial cells of the BBB is controlled by Mfsd2a. The lipid composition of the plasma membrane which is highly enriched by DHA impairs the formation of caveolae vesicles. In the absence of Mfsd2a, transcytosis across the endothelial cell increases. BBB: blood-brain barrier, DHA: Docosahexaenoic acid, LPC: Lysophosphatidylcholine.

               The researchers analyzed brain metastases using patient-derived xenograft (PDX) which is a tissue graft from a donor of a different species from the recipient. In this case mouse models were used to study signaling pathways involved in disruption of the intratumoral BBB. Cells were initially grown in serum-free media, with the intention of relieving the effects of substances such as growth factors on the cells. Cultured metastatic tumor cells grew as neurosphere-like spheroids in serum-free media and expressed epithelial markers including E-Cadherin and β-catenin. Specific patterns of localization and expression of Mfsd2a in brain endothelial cells suggested a functional role that was further investigated. Since intratumoral blood vessels showed abnormal BBB properties, the authors analyzed the expression of Mfsd2a in metastatic tumors and compared these results to the opposite, non-injected hemisphere. The results evidenced a statistically significant decrease of Mfsd2a protein expression in vascular endothelial cells from brain metastases originating from primary breast cancer.

               Importantly, the results containing reduced numbers of perivascular astrocytes strongly correlated with lack of Mfsd2a expression in brain metastasis endothelial cells. The data supports the notion that endothelial cells of brain metastases are in contact or are associated with pericytes but show diminished interactions with perivascular astrocytes. As determined previously, the presence of pericytes was confirmed to be required for the expression of Mfsd2a in the brain endothelial cells. The analysis of previous microarray data of pericyte‐deficient mouse models (Armulik et al., 2010) demonstrated a significant decrease in Mfsd2a expression in pericyte‐deficient animals (Ben-Zvi et al., 2014). This data can be corroborated with reduced expression of Mfsd2a. Further, astrocytes have been known to secrete cytokines and growth factors that modulate BBB properties in the brain vascular endothelium. For this reason, the researchers examined the influence of conditioned media taken from primary mouse brain astrocytes or human astrocytes on the expression of Mfsd2a in low passage. This means that the cells were not passed or split more than five times in order to keep mutations and selective pressures at a minimum to control for mutations. This demonstrated that both mouse and human astrocytes secrete factors that promote the expression of Mfsd2a mRNA in HBMECs.

               It is also known that Mfsd2a is a transporter for DHA when it is conjugated to lysophosphatidylcholine (LPC) in circulation, as described by the pathway in Figure 2. Thus, the effects of astrocyte conditioned media on Mfsd2a-dependent uptake of nitrobenzoxadiazole-LPC (NBD-LPC) were examined using a fluorescent tracer, revealing an increased uptake of NBD-LPC by HBMECs treated with astrocyte-conditioned media compared to a control non-conditioned media. The astroglial-derived factors that positively regulate the expression of Mfsd2a were determined by treating HBMECs with different cytokines with known effects on endothelial barrier properties, such as VEGF, bFGF/FGF2, and TGFβ1. After treatment, levels of Mfsd2a gene expression were measured using qRT-PCR. It was concluded that both bFGF and TGFβ1 have the ability to induce the expression of MFSD2A in HBMECs, whereas VEGF had an inhibitory effect on MFSD2A gene expression.

Figure 2. The proposed mechanism for Mfsd2a‐mediated LPC transport across the plasma membrane is Na dependent. Na binds to Mfsd2a and LPC inserts itself into the outer membrane and diffuses laterally into the hydrophobic cleft of Mfsd2a.
this causes a conformational change in Mfsd2a to an inward‐open form while the zwitterionic headgroup of LPC is inverted within the transporter (Quek et al., 2016). Na: sodium.

               Finally, to evaluate a direct effect of DHA on metastases, the authors treated cultured primary lung and breast brain spheroids with DHA. They observed decreased growth and survival of tumor cells treated with DHA compared to controls. Additionally, DHA treatment positively impacted the growth and survival of HBMECs. Therefore, the researchers conclude that loss of Mfsd2a in metastatic tumor endothelial cells leads to decreased uptake of essential fatty acids, specifically DHA, which promotes tumor growth and survival in the brain microenvironment.

               Mediating the transport of DHA is not the only function, however. In addition, Mfsd2a suppresses caveolin-dependent transcytosis. Increased vesicles in Mfsd2a‐deficient mice have been found to be positive for caveolin‐1 (cav‐1) which is the mandatory protein coat implicated in the formation of caveolae vesicles from the plasma membrane (Ocak et al., n.d.). DHA was also previously shown to displace cav‐1 from the plasma membrane, decreasing the formation of caveolae vesicles in its presence. This was demonstrated using a genetic deletion of murine Mfsd2a leading to enhanced transcellular transport and breakdown of the vascular endothelial barrier in the brain and retina. The research shows that Mfsd2a expression as well as its transport functions are down-regulated in the metastatic brain tumor vascular endothelium and can be explained by the absence of astrocytes that use TGFβ1 and bFGF signaling to maintain expression of Mfsd2a in cerebral endothelial cells.

               Lipid transport pathways of Mfsd2a pay a critical role in the regulation of permeability and in the maintenance of the integrity of the BBB by acting as a suppressor on caveolae‐mediated transcytosis in the endothelial cells of the CNS vasculature. Therefore, loss of MFSD2A promotes metastatic tumor growth and survival in the brain microenvironment by altering DHA transport and metabolism. Mfsd2a is a novel LPC transporter selectively expressed in the endothelial cells of the CNS and provides impactful contributions to the formation, functioning, and maintenance of the BBB. Looking ahead to the future, this pathway can possibly be harnessed for pharmaceutical delivery across the BBB. Changes in Mfsd2a expression levels following different types of brain injury may be unique to the pathology, which will be an important factor to consider while creating specific targeted therapeutic strategies. Nonetheless, large numbers of in vivo and in vitro studies are warranted in order to corroborate the practicality and applicability of pharmacologic strategies based on the modulation of BBB permeability via Mfsd2a. Restoring DHA and or its metabolites to the tumor microenvironment may serve as a treatment option for patients with metastatic brain cancer.

References:

1. Ocak, P.E., Ocak, U., Sherchan, P., Zhang, J.H., Tang, J., n.d. Insights into major facilitator superfamily domain-containing protein-2a (Mfsd2a) in physiology and pathophysiology. What do we know so far? J. Neurosci. Res. 0. https://doi.org/10.1002/jnr.24327

2. Pericytes regulate the blood–brain barrier | Nature [WWW Document], n.d. URL https://www.nature.com/articles/nature09522 (accessed 2.11.19).

3. Structure and function of the blood–brain barrier – ScienceDirect [WWW Document], n.d. URL https://www.sciencedirect.com/science/article/pii/S0969996109002083?via%3Dihub (accessed 2.11.19).

4. Tiwary, S., Morales, J. E., Kwiatkowski, S. C., Lang, F. F., Rao, G., & McCarty, J. H. (2018). Metastatic brain tumors disrupt the blood‐brain barrier and alter lipid metabolism by inhibiting expression of the endothelial cell fatty acid transporter Mfsd2a. Scientific Reports, 8(1), 8267. https://doi.org/10.1038/s41598-018-26636-6

5. Nguyen, L. N., Ma, D., Shui, G., Wong, P., Cazenave‐Gassiot, A., Zhang, X., … Silver, D. L. (2014). Mfsd2a is a transporter for the essential omega‐3 fatty acid docosahexaenoic acid. Nature, 509(7501), 503–506. https://doi.org/10.1038/nature13241

8 thoughts on “Lipids on the Mind

  1. Hi Alyssa! Thanks for sharing this article! Mfsd2a seems to have a multitude of functions. In the last paragraph, you mentioned that DHA could serve as a possible treatment for metastases to the brain, which makes sense given the experiment that showed that DHA-treated tumor cells showed decreased growth and survival. However, if Mfsd2a expression is decreased in tumor cells and is responsible for DHA transport, would just supplementation of DHA be enough as a treatment? Or could it be done in conjugation with one of the cytokines or another compound that stimulates expression of Mfsd2a?

    1. Hello!! As I mentioned in response to Kelly, the research that the authors put forth points to a loss of Mfsd2a in metastatic tumor endothelial cells leading to decreased uptake of essential fatty acids, specifically DHA, which may promote tumor growth and survival in the brain microenvironment, but decreased Mfsd2a expression also correlates to increased permeability of the intratumoral BBB. The only proliferative studies conducted focused on the addition of DHA and I think should absolutely be conducted in conjunction with cytokine induction. The only this is is that (as I mentioned above), inducing cytokines can often result in inflammation which is particularly dangerous in the brain and may not be the best course of action; in that case, it would necessary to find another method of upregulating Mfsd2a. It would be interesting to compare the proliferative ability of metastatic cells in the presence of the upregulation of cytokines compared to the addition of DHA or combining both targets in one model. I mentioned this in Kelly’s response, as well, but I would also like to know if an alteration in the intratumoral BBB permeability could lead to more effective delivery of drugs targeting metastases that are superior to treatments with DHA.

  2. It is interesting how the authors challenged this paradigm: “Other studies have reported increased levels of omega-6 fatty acids, especially arachidonic acid (AA) in response to reduced levels of DHA” (6) by proving that both the levels of DHA & AA are decreased in tumor cells, then showing that the ratio of omega-6 to omega-3 is increased. This provides evidence for potential treatment of brain tumors through exposure to high levels of DHA, but my question is: how does additional DHA get through the BBB if the tumors have already inhibited DHA’s transporter Mfsd2a? I could be missing something from the paper, but what other steps would have to be taken in order to get DHA through this barrier?

    1. It is acknowledged that a prior study reported increased levels of omega-6 fatty acids, especially arachidonic acid (AA) in response to reduced levels of DHA in normal brain tissue. This study, conducted in 2014, has been up-cited more than 300 times! While Tiwary et al., 2018 was surprised to learn that levels of AA-conjugated phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS) were reduced in metastatic tumor tissue versus the normal brain, suggesting an overall down-regulation of essential fatty acid metabolism in brain metastases, they did observe an overall increase in the ratio of AA (omega 6) to DHA (omega 3) conjugated PE, PC and PS in the brain metastasis. This source also reports that Mfsd2a did not transport unesterified DHA or any other fatty acids, rather, LPC DHA could be transported in a concentration-dependent manner. Nguyen et al., 2014 also notes that Mfsd2a-knockout mice had a reduction in brain uptake of LPC [14C]DHA and LPC [14C]oleate by 90% and 80%, so there must be another mechanism in brain metastases that remains unidentified.

      The 2018 data reveals a decrease in overall levels of AA conjugated PC, PE and PS in mouse models of breast cancer brain metastasis, which agrees with other studies reporting an increased ratio of omega-6 to omega-3 fatty acids in pathological conditions including cancer. Further, down-regulation of Mfsd2a expression in the vascular endothelium of brain metastasis hints that that tumors fail to transport and metabolize DHA, possibly for selective growth and survival advantages. The authors hypothesize that DHA is able to suppress metastatic malignancy either by “inhibiting early seeding and growth of neoplastic cells in the brain microenvironment prior to their eventual inhibition of Mfsd2a and DHA transport,” but this remains to be elucidated and I have not found any literature that explains this finding.

      Reference: Nguyen, L. N. et al. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 509, 503-+, https:// doi.org/10.1038/nature13241 (2014)

  3. Alyssa,
    It is very compelling how the article makes a string of connections from low expression Mfsd2a proteins to increased development of certain metastatic cancers in the brain. The findings suggest cytokines bFGF/FGF2 increase Mfsda2A protein expression which facilitates DHA transport across the blood brain barrier which in turn decreases survival of cancer cells. In one experiment the researchers introduce DHA into cancer cells in another the researchers introduce cytokines to increase Mfsda2A expression which would in turn increase DHA. In both experiments the researchers observe improvement of disease course. Within this chain where do you think the is the best/most efficient target for treatment of these cancers would be?

    1. Kelly!! Sorry in advance for the long response.

      There are multiple levels to this question. The research was prompted by a genetic deletion of Mfsd2a protein in mice that led to impaired DHA transport and a lower presence of lipid metabolites. Firstly, breast cancer brain metastasis models showed blood vessel structures lacking a CD31-expressing lining endothelium, implicating CD31 in the integrity of intratumoral vasculature. From this, quantitative data demonstrated a significant increase in permeability of blood vessels in this tissue, and Mfsd2a protein expression was decreased in these in vascular endothelial cells. This mechanism could potentially be exploited for administration of antitumoral treatments. I would then wonder if this method would be even more beneficial than DHA treatments.

      Another correlation was made between reduced numbers of astrocytes and a lack of Mfsd2a expression in brain metastasis endothelial cells since both mouse and human astrocytes secrete factors that promote expression of Mfsd2a mRNA in HBMEC. Upon further investigation, the astrocytes secrete factors that promote expression of Mfsd2a mRNA in HBMEC. As you mentioned, cytokines with known effects on endothelial barrier properties were used including bFGF/FGF2 and TGFβ1 and both induced the expression of MFSD2A in HBMECs. Because TGFβ signaling stimulated Mfsd2a expression in the cultured endothelial cells the authors continued by analyzing Mfsd2a expression including the endothelial cell-specific inhibition of TGFβ signaling that is actually present in mice resulting in a less promising production of Mfsd2a mRNA.

      Anyways, because of the down-regulation of Mfsd2a expression in the endothelium of brain metastasis, the authors hypothesized that tumors fail to transport and metabolize DHA, possibly for selective growth and survival advantages, and in fact, NBD-LPC uptake was significantly decreased in tumor tissue compared to the normal brain. The researchers further noted decreased growth and survival of tumor cells treated with DHA compared to the control.

      All of this information points to a loss of Mfsd2a in metastatic tumor endothelial cells leading to decreased uptake of essential fatty acids, specifically DHA, which may promote tumor growth and survival in the brain microenvironment, but also correlates to increased permeability of the intratumoral BBB. The only proliferative studies conducted focused on the addition of DHA. Inducing cytokines can often result in inflammation which is particularly dangerous in the brain and may not be the best course of action. It would be interesting to compare the proliferative ability of metastatic cells in the presence of the upregulation of cytokines compared to the addition of DHA or combining both targets in one model. Finally, I would like to know if an alteration in the intratumoral BBB permeability could lead to more effective delivery of drugs targeting metastases that are superior to treatments with DHA.

  4. Alyssa, amazing article and spotlight! In your spotlight, I learned Mfsd2a selectively transports omega-3 cross blood-brain barrier, however, in the article, it seems like authors didn’t pay too much attention to its selectivity (maybe I missed something). Anyway, I think the selectivity of Mfsd2a to DHA is a good point to dive in by modifying the Mfsd2a expression.

  5. So this article is fascinating and posses serious implication for future research into the study of brain cancer/ metastasis. So if Mfsd2a regulates the uptake of essential fatty acids like DHA which can slow the growth of malignant cells is it possible that coupling a small anti-tumor molecule to DHA allow for greater cancer cell destruction. Or would it be more useful to un-inhibit Mfsd2a thus increasing the BBB permeability allowing for new novel drugs better access to the brain and the tumors?

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