MK-4 reduces the production of FGFR3 in liver cancer cells. Can this finding result in a new pharmacological approach for achondroplasia?

Please, keep in mind that the content of this article has been adapted for the non-technical reader and is only a brief review of the subject.

MK-4, also known as menatetrenone, inhibits FGFR3 production in cancer

In 2009, a Japanese group, studying the effects of MK-4, a menaquinone (see more information about below), in a model of liver cancer (hepatocarcinoma) found that MK-4 helped to inhibit cancer cell proliferation (multiplication) and survival because it reduced the production (expression) of fibroblast growth factor receptor type 3 (FGFR3) and stimulated the expression of a protein called p21. (1)

If you have been reading the articles published here you may already know that the usual role of FGFR3 is as growth promoter in the vast majority of cells inside our body. That’s the reason why FGFR3 is important in certain types of cancer, such as multiple myeloma and bladder cancer, which use FGFR3 properties to grow. A relevant cell exception known for this growth promotion role of FGFR3 is exactly the chondrocyte. For chondrocytes of the cartilage growth plate, FGFR3 is a growth brake, modulating the growth speed in concert with other local or systemic growth promoters. In achondroplasia, as FGFR3 is working too much, bone growth is impaired.

MK-4 inhibits FGFR3. Is this good news?

I mentioned that in the paper by Cao et al. (1) another protein was stimulated, the p21. This is relevant information because p21 is a cell proliferation controller, one of the proteins which regulate the process by which the cell multiply, called mitosis. In mitosis, p21 is a brake. Well, for us who are thinking in a pharmacological solution to treat the bone growth impairment in achondroplasia, the last thing we would want would be to have a drug further disturbing cell growth.

And then? Should we, because a possible undesired effect, just throw away this interesting information about a compound that could reduce the influence of FGFR3 in achondroplasia?

I think this is not the case and the reason is simple: from a scientific point-of-view one should only say a phenomenon is happening or not by testing the hypothesis. In a parallel with the distinct functions proteins may exert in different cells and tissues in the body, drugs may exert their actions in different cells in different ways. It is worth to check what happens with a chondrocyte exposed to MK-4.

And what if we found another evidence for a possible MK-4 effect in bone growth?

Mice exposed to MK-4 grew more than those unexposed

Japanese researchers have been giving strong attention to menaquinones, specially, MK-4 and MK-7 because of their already known actions in bone health (see below). Although the biological actions of menaquinones are recognized, the clinical impact of them in human health is still under debate (see below). There are tens of studies exploring the actions or effects of MK-4 supplementation in osteoporosis and bone health coming both from Western and Eastern countries. One paper published in 2011 in the journal Bone (2) sought to compare the long term effects of MK-4 and vitamin K1 in the bones and health of young growing mice. In general, bones in treated animals were stronger than those of the controls. However, one finding in this study called the attention: the treated mice were higher (or longer) than the control animals: they grew more. Could be this finding a result of MK-4 effect in FGFR3? Soon after the paper was published I wrote a letter (3) to the journal asking if the investigators would have tested the expression of FGFR3, one of the genes expressed in the growing bones. Unfortunately, the investigators did not study the growth plate or FGFR3 in their animals. By the other side, in a more recent study (4), another group of Japanese researchers did not demonstrate growth differences between MK-4 treated mice compared to controls, although the dose used was lower than in the study by Sogabe et al.  

Nevertheless, we have some small evidence from two different studies about the potential role MK-4 would have in bone growth, maybe exerting an action in FGFR3.

Now, it is time to learn a bit about MK-4 and the menaquinones. This will be useful to set adequate expectations about this class of compounds in the context of achondroplasia.

MK-4 is part of the Vitamin K family

                                  

Vitamin K (VK) was first recognized as an essential cofactor for the normal coagulation process because it participates in the assembly of a protein called prothrombin. You can find diagrams and more detailed information about the vitamin K family following this link.

In summary, the VK family is divided in two main groups. The VK1 is called phylloquinone and is the most known form of the vitamin. Its main dietary source is the leafy green vegetables like spinach and broccoli. The other main group, called VK2, is formed by the menaquinones, which are also named MK-n (MK-4 to MK-13), where “n” stands for the number of isoprenol moieties located at the position 3 of the napthoquinone ring (the core of the molecule). The menaquinones are produced by bacteria, so they are found mainly in fermented food, like cheese and fermented soy bean (natto, a Japanese specialty) (5). While VK1 is stored preferentially in the liver, the VK2 species (menaquinones) are found mainly in extra-hepatic tissues, such as bones (6,7).

In the last decades a mounting volume of information has been accumulated about other metabolic functions in which VK is involved, with the identification of an increasing number of proteins that depend on the presence of VK to become active. These so called VK-dependent proteins (VKDP) are in a way or another associated to the handling of calcium in body tissues such as the bones, cartilage and the blood vessel walls. The most studied of these proteins is osteocalcin. Osteocalcin is a calcium binding protein and it is likely to have an important role in contributing to the strength of the bones and to balance the function of osteoblasts and osteoclasts, the cells that respectively build and absorb the bone structure (8).

Menaquinones help to keep bone health

Many studies about the menaquinone's properties in the bone come from Japan, where there has been intensive research with MK-4 and with MK-7. Moreover, the Japanese have been recommending the use of VK2 to prevent and treat osteoporosis for more than a decade (9).

Besides osteocalcin, there is another important VKDP called Matrix Gla-rich Protein (MGP), which is pivotal for cartilage health and development. MGP is produced by chondrocytes, the cells living within the cartilage. It seems that MGP captures calcium ions in the cartilage and vessel walls, helping to regulate calcium in those tissues where this ion plays important metabolic roles. In the growth plate, MGP is thought to have some role in delaying the calcification of the matrix, the material within lie the chondrocytes. MGP is particularly more abundant in the hypertrophic layer of the cartilage growth plate (10). MGP is also produced by cells in blood vessels. Mice lacking MGP have early arterial calcification and short stature due to inappropriate calcification of the cartilage growth plate (11). So you see, it seems that the menaquinones probably have more roles in bone development than usually supposed. As mentioned above there is a long term debate regarding the actual usefulness of menaquinones to prevent or treat osteoporosis. Studies coming from Japan have been showing positive outcomes while studies performed in Western countries have not been describing a positive action of menaquinones (MK-4 or MK-7) in bone strength. Looking at the literature it appears that what has been difficult to define is how much important is the presence of these compounds for bone health.

MK-4 inhibits FGFR3. How can this be tested in achondroplasia?

MK-4 is being tested in a vast number of mouse/rat models, in several different conditions, to analyze its ability to increase bone density or to prevent and treat osteoporosis. Tests are being performed in doses thousands of times larger than the VK nutritional doses recommended by Health Agencies. This happens because the daily dose of VK needed to achieve a good control of coagulation is very small. There is no standard dose set for other biological functions of VK. In Japan, however, for osteoporosis the dose used, 45mg/day, is far larger than that of the diet recommendation (~50 to 120 mcg/day, depending on the country) (7). These low amounts of VK1 are sufficient to keep the coagulation process normal but it is insufficient to activate in full the other VKDPs.

Testing MK-4 is a matter of will

Since the paper by Cao et al. (1) was published, I wrote to several different investigators around the world showing the study results, asking if they would be interested in testing the hypothesis that MK-4 could be used in achondroplasia but the answer varied from a nice or testing it is too expensive to this is a vitamin, or simply there was no response at all.


Part of the difficulty to have a new compound tested for a given situation is true lack of funding. The experiments are really expensive. But, in the case of MK-4, I think there is also a bit of prejudice interfering in the decision to check its potential. You see, MK-4 is called a vitamin, so how can you believe a vitamin could treat a genetic condition? The thing here is that nobody should expect that, if MK-4 ends to be useful in our context, the intention is to just give nutritional doses of the compound and a miracle will take place. Not at all, if MK-4 could be used for achondroplasia, the expected dose needed would be very, very high, in what is called a pharmacological use of the compound. In other words, we would be talking about a real drug not a vitamin. That's why you should not buy MK-4 at will and start giving it to a child based just on what is written here. I am very far indeed from recommending this to anyone.

First things first

Some fundamental steps are needed before start to think in using MK-4 in individuals with achondroplasia.

The first one is to show how MK-4 works in mutated chondrocytes. We need to see if FGFR3 inhibition occurs in chondrocytes in the same way it does in liver cancer cells. Also important, we must see if there are other important proteins being influenced by MK-4 (for instance, p21).

The second step is to see the effect of MK-4 in animal models of achondroplasia. From investigating the dose needed to achieve any result to monitoring safety parameters, this step is crucial to confirm if MK-4 could be used in affected individuals. Although MK-4 has been showing to be very safe in animals and humans, safety should be kept as a main concern due to the expected very large doses which would be needed to see any effect compared to the nutritional ones. It is worth to mention that menaquinones are thought to be safe. Several studies showed that even with doses hundreds of times larger than the dietary recommended ones, no definite relevant adverse events are reported (9).

Once tests in laboratory and in appropriate models have demonstrated that MK-4 is active against FGFR3 and rescued the growth in those models, we will reach the final step which is to test MK-4 in humans.

If such effect is observed, a new, simple, easy-to-use, probably safe, costless therapeutic alternative would be readily available for children bearing FGFR3 mutations. Moreover, if this alternative is valid, the therapy should be started as early as possible and should be kept until adulthood.

What we need right now is to have investigators engaged in proving, or discarding, this hypothesis. There is a new, wide road waiting to be explored. Who will take this job?

References

1. Cao K et al. Vitamin K2 downregulates the expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma cells. Hepatol Res 2009; 39(11):1108-17.

2. Sogabe N et al. Effects of long-term vitamin K(1) (phylloquinone) or vitamin K(2) (menaquinone-4) supplementation on body composition and serum parameters in rats. Bone. 2011 May 1;48(5):1036-42.

3. Kaisermann MC. Vitamin K family effects on bone growth. Bone 2011; 48(6):1427.

4. Matsumoto T et al. Effects of vitamin K on the morphometric and material properties of bone in the tibiae of growing rats. Metabolism. 2012;61(3):407-14. 

5. Schurgers LJ, Vermeer C. Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations. Haemostasis 2000; 30: 298–307.

6. Shearer MJ, Newman P. Metabolism and cell biology of vitamin K. Thromb Haemost 2008;100: 530–47. (free)

7. Shearer MJ, et al. Vitamin K nutrition, metabolism, and requirements: current concepts and future research. Adv Nutr. 2012;3(2):182-95. 

8.Gundberg CM et al.Vitamin K-dependent carboxylation of osteocalcin: friend or foe? Adv Nutr. 2012 Mar 1;3(2):149-5.

9. Iwamoto J et al. High-dose vitamin K supplementation reduces fracture incidence in postmenopausal women: a review of the literature. Nutr Res 2009; 29: 221–8.

10. Dan H et al. The role of matrix gla protein in ossification and recovery of the avian growth plate. Front Endocrinol (Lausanne). 2012;3:79. Epub 2012 Jul 10. (free)

11. Luo G et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 1997;386(6620):78-81.