July 17 2010 post. Thanks to a comment left by Joao on my previous post, I was able to read a rather exciting bit of news this morning. In a nutshell, a team of Swedish/Belgian researchers has identified, and made a list of!, a group of genes that are “silenced” in myeloma cells but NOT in normal plasma cells. This “silencing” process, which is carried out by a group of proteins called Polycomb repressors or PcGs, may have a lot to do with the uncontrolled growth and “self-renewal” of myeloma cells. More testing and research are needed, of course, but I already consider this a verrrrry important discovery (…incidentally, here is the link to the Science Daily article: http://tinyurl.com/37mbzw5).

The full study is available for free online (oh, I do love freebies!): http://tinyurl.com/3y8errz All you have to do is click on “Download PDF.” The study is VERY technical: now you can’t say I didn’t warn you!!! Since, as you know from my previous post, Florence and the rest of Italy are smack in the middle of a horrendous heatwave, I didn’t and don’t have the energy to read the whole shebang in one sitting, but here are a few interesting excerpts I took from the Discussion part of the study:

1. The silenced genes were found also in MGUS, which means that this “shutting up” process might be an early event during MM development.

2. The “silencing” was also more pronounced in the advanced stages (ISS stage III compared to stage II and I) of MM progression. Huge implications, here…

I need to read this study in full and pay particular attention to the part discussing the similarity between cancer and stem cells. As you know, I am a strong supporter of the myeloma stem cell theory…This study suggests, however, that myeloma stem cells (a small subpopulation of clonogenic CD138 negative B cells) probably do not have an impact on the underexpressed gene profile, when analyzing the tumor bulk. And this profile seems to be a general feature shared by a large proportion of the MM cells. 

July 19 2010 post. Well, here, finally!, are my notes on the Discussion part of the “silencing” study (see my July 16 post). I was forced to skip through the bits that I didn’t really understand..and would therefore welcome comments/corrections from anyone who has a scientific mind/background. Thank you!

Okay, let’s dive right in…the researchers point out that multiple myeloma doesn’t have a common origin that would help provide a universal MM drug target. While it is true that previous studies have identified important signalling pathways (does the infamous NF-kappa B ring a bell?), previous gene expression profiles to date have mainly focused on subdividing MM into genetic entities for prognostic or therapeutic purposes. The aim of this study was instead to find a common denominator for myeloma development and tumor maintenance. A high goal indeed…

The researchers identified a set of “underexpressed” genes located inside MGUS and MM cells. But there is also a second group of genes, a pesky group of hooligans called the Polycomb Group, or PcG for short. The Polyhooligans (=my invention) go after and “silence” all the good guys, I mean, all the good genes. And, as I mentioned a few days ago, this “silencing” activity becomes more frenetic in more advanced stages of myeloma. Bad, bad stuff…

Before going on, let’s take a quick look at the term “underexpression.” In our particular case, as I understand it!, this means that a smaller amount of a particular gene is present in a myeloma cell when compared to a normal plasma cell. Underexpressed = decreased. And here is an example from the study: the researchers found that the CIITA (=Class II transactivator…ah, don’t even ask!) gene was on their list of “silenced genes.” Why am I mentioning this? Because the underexpression of CIITA enables a myeloma cell to dodge the immune system…

Another important finding is that the Polycomb repressor genes appear to be a common feature among the tumor cells, rather than representing a specific subpopulation. The researchers indicate that Epigenetic gene silencing is an attractive drug target due to the fact it can be reverted. So the researchers tested two chemical inhibitors with impossible-sounding names but thankfully-short acronyms: DZNep and LBH589. And yes, these two thingamajigs were able to rouse a selected group of the slumbering genes, including our newest friend, Ms. CIITA.

The chemical substances had other beneficial effects as well. Here is one example: EZH2 is a protein that can become overexpressed (=the opposite of underexpressed…) in multiple myeloma. This is bad, clearly, since the ”going crazy” of EZH2 leads to the proliferation and survival of myeloma cells. Well, the above-mentioned chemicals decreased the amount of EZH2 inside the myeloma cells. This is not that surprising, since both chemicals do the same in AML and breast cancer cells…

Another positive effect: In addition to the reactivation of the Polycomb-target genes both DZNep and LBH589 reduced viability and induced apoptosis in two MM cell lines. Yes, that means that, in addition to waking up our sleeping beauties, these two chemical inhibitors exterminated a number of myeloma cells…

The researchers tested LBH589 in vivo (=mice), with the following result: Treatment with LBH589 significantly decreased tumor load and levels of paraprotein AND increased overall survival. They also found that it can overcome drug resistance in MM cell lines and cells from patients with refractory disease, so it might be useful in combination with other drugs.

[Quick aside: the study mentioned that LBH589 was being tested right now in myeloma clinical trials. So off I went to the Clinical Trials website where I found eight clinical trials in the process right now of recruiting relapsed/refractory myeloma patients for an LBH589/Panobinostat trial in combination with other drugs. Interesting. Okay, back to us, now…]

Previous studies have shown that PcGs are involved in the development of other types of malignancy. And they are important in embryogenesis, which is the process whereby an embryo is formed and develops. So they are not ALWAYS violent, bad guys…just in cancer. This bit will clarify, I hope!, the following statement (where “epigenetic mark” = PcG): The fact that an epigenetic mark maintaining self-renewal in embryonic stem cells is found in cancer supports the hypothesis that cancer cells share features with normal stem cells. This is significant because, based on the data, the stem-like features of myeloma cells seem to be crucial for their survival and self-renewal.

In conclusion, the above-mentioned “common denominator” would be the group of silenced/underexpressed genes = the good guys. Then we have the bad guys, the Polyhooligans, who spend all their time lurking in the darkest shadows of our myeloma cells and whose sole purpose in life is to hunt down and knock out a bunch of friendly, nonviolent genes that are then no longer able to function properly, as they would under normal circumstances (in normal plasma cells, that is). This “silencing” process enables myeloma cells to proliferate and survive, completely undisturbed… Well, at any rate, that is how I interpret this incredibly complicated process (and text!)…right or wrong…

Is there anything we can do to stop the Polyhooligans? Well, I hope to have a few suggestions in the next few days…First, though, I have to read the studies…so stay tuned…

Helpful information on the Polyhooligans: http://tinyurl.com/2ecv29s

July 26 2010 post. I began my Polycomb repressor gene quest with this study: http://tinyurl.com/2vboddk, which examines the activity of the Polyhooligans in Hodgkin’s lymphoma cell lines. As you can see, the abstract is a bit on the technical side…This will give you an idea of the kind of stuff I have been reading lately…Okay, now for the relevant part of the full study…

As I mentioned in my July 19 post, the Polyhooligans are bad only when it comes to cancer cells. And, since it is their cancer cell goings-on that interest us, I will skip all the parts in the study concerning body formation, haematopoiesis and cell cycle control.

The full study tells us that a couple of the Hodgkin’s lymphoma cell lines were treated with curcumin and that a decrease in the level of MEL18 expression after curcumin exposure was […] observed in both lines. The cells also showed inactivation of NF-kappaB after being exposed to curcumin.

Another interesting excerpt: when TNF-alpha, a well-known activator of NF-kappaB, was added to these cell lines, the activity of MEL18 increased. These findings support the relationship between NF-kappaB activity and the expression of MEL18...I hope this part is clear enough, since I think it’s amazing. I mean, curcumin inhibited one of the Polyhooligans, the one called MEL18, which seems to be closely connected to NF-kappaB, our old nemesis (=small world, eh…). Fantastic!

Well, this is just the beginning…

July 28 2010 post. I read a 2005 “Oncogene” study (full text available for free: http://tinyurl.com/3y4fclq) for one, and only one!, reason, which we will get to in a second. Let’s see…I could simply write that, according to this study, EZH2, which stands for “enhancer of zeste homolog 2? (ah, don’t ask!), is a pesky Polycomb repressor gene that is hyperactive, in a bad sense!, in myeloma as well as other types of cancer…but no, that is not enough. We must examine a few excerpts that will help us understand the connection with the second study discussed in this post…

The “Oncogene” study tells us that Normal bone marrow plasma cells do not express ezh2; however, gene expression is induced and correlates with tumor burden during progression of this disease. What this means is that EZH2 is present only in myeloma plasma cells, not in normal plasma cells. Furthermore, the hyperactivity of EZH2 increases as myeloma progresses (a bad thing, clearly): that is basically what “correlates with tumor burden” means.

These researchers discovered that EZH2 is closely linked to IL-6, which is perhaps the most written-about myeloma interleukin (I have written a lot about it, too). Just as a reminder: IL-6 is involved in a host of myeloma-centered activities, including progression and proliferation… If you would like to read more about the link between the Polyhooligan and IL-6, please have a look at the above-mentioned study…

Let’s get back to the point, now. When the EZH2 Polyhooligan becomes overly active and hopped up (=overexpressed), myeloma cells are as happy as clams and grow like mad; the reverse occurs, however, when this Polyhooligan is inhibited…that is, myeloma cells stop growing when EZH2 is inhibited. This is a very VERY important point. Uhm, okay, I won’t go into the part about “mutant N- or K-ras gene,” except to say that EZH2 is involved in that process, too. EZH2 is a very bad, BAD boy!

The study tells us that this super-active Polyhooligan is very much involved in metastatic prostate cancer and aggressive breast cancer. But it adds a titbit that is important for us myeloma folks to know: ezh2 was found to be one of the 30 genes that could distinguish between normal PC and MGUS (MM1) and aggressive myeloma (MM4). Eh…

As mentioned above, sorry for the repetition!, this particular Polyhooligan can be found only in multiple myeloma cells, not in normal bone marrow plasma cells. And EHZ2 is also much more active and wild in Stage 4 than in Stage 1. Its activity is therefore associated with myeloma progression.

Well, the researchers discovered that the growth of myeloma cells was significantly affected when EZH2 was blocked (they used a specific synthetic EZH2-inhibitor known as siRNA). They conclude: This study also suggests that EZH2 is required for MM cell growth since all MM cell lines studied were significantly growth inhibited upon EZH2 siRNA treatment. Wow, this sentence is of immense importance…

I could go on and on and on, but I don’t want to beat my point to death, especially since you can read the full study online for free. All we really need to keep in mind is that this particular Polyhooligan is linked to myeloma cell proliferation and must be stopped. But how?

I am sure that by now most of you have already guessed that our best buddy, curcumin, inhibits EZH2. Ah yes. Now, even though the amazing anti-this-and-that-and-everything properties of curcumin shouldn’t surprise me anymore (after all, I have spent the past four years researching this topic!), I have to admit that I was indeed surprised to find this July 2010 study on the extraordinary effects of curcumin on EZH2 and metastatic breast cancer cells: http://tinyurl.com/3y5plyq Before I forget, I would like to thank a blog reader/friend (thanks!) for sending me the full text…

The basic information is in the abstract: curcumin inhibits EZH2 in a highly metastatic breast cancer cell line. Please pay attention to the underlined (by moi!) items mentioned in this sentence: “Further investigation revealed that curcumin-induced down-regulation of EZH2 through stimulation of three major members of the mitogen-activated protein kinase (MAPK) pathway: c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase.”

These three kinases (=a kinase is merely a type of protein, by the way) are important in myeloma, too. Quelle surprise…not. For more information, please have a look at this study: http://tinyurl.com/35rokpa (hmmm, at some point I should probably devote a post to galectins, which I’d never heard of before…).

Let’s dive right into the breast cancer-curcumin-EZH2 study. It almost immediately refers to the 2004 study (see my July 26 post on curcumin and MEL18), which suggests that curcumin might be involved in the regulation of PcG proteins and related signalling pathways. 

Further on we read: It is known that the enhancer of zeste homolog 2 (EZH2) is a critical component of PcG proteins, and that EZH2 plays an important role in cell proliferation and cell cycle regulation. The overexpression of EZH2 has also been reported to be linked to invasive growth, aggressive clinical behavior and poor prognoses of breast cancers.

These researchers discovered that curcumin inhibited the proliferation of a particularly aggressive breast cancer line in a dose- and time-dependent manner. This inhibition was significant when compared to untreated control cells, and the dose used was a non-toxic one. (For those with a scientific inclination: cell development was arrested in the G1 phase.)

The Discussion part contains a lot of food for thought and is not too difficult to follow (in fact, it is also a bit repetitive…): Recently, polycomb group proteins have been suggested as candidates for molecular targets of breast cancer treatment; and a critical component of the polycomb group proteins is EZH2. It has been found that EZH2 expression is significantly up-regulated in invasive breast carcinoma and cancer metastases, and that overexpression of EZH2 is associated with poor outcomes in breast cancer patients.

The researchers observed that the activity of EZH2 decreased as soon as curcumin managed to block the proliferation of these breast cancer cells. They therefore surmised that these two events might be connected–curcumin stops the proliferation of the breast cancer cell line, at least in part, by the down-regulation of EZH2 expression and its activity.

The researchers also mention a recent study showing that curcumin can activate MAPK signaling pathways to induce cell apoptosis in HCT116 cells and cisplatin-resistant human ovarian cancer cells. I have a good reason for mentioning this part: the activation of this particular pathway plays an important role in the annihilation of myeloma cells, too (a number of studies on PubMed confirm this point, including the above-mentioned galectin study).

When the MAPK pathway was inhibited, so was the curcumin-induced down-regulation of EZH2 expression, the researchers found. Therefore, the activation of MAPK seems to help curcumin to have an impact on the activity of EZH2. Sounds like gibberish, I know. But, in a nutshell, curcumin activates the MAPK signalling pathway AND decreases the hyperactivity of EZH2 in metastatic breast cancer cells, thus inhibiting their proliferation…wowiezeeewee!

The researchers conclude that curcumin, known to be pharmacologically safe, could profitably be used to target EZH2 for breast cancer treatment.

Indeed…not just for breast cancer, it would seem…based on my own findings…

July 30 2010 post. Hah, you didn’t think I had set the Polyhooligans aside just yet, did you?    Well, as we know from my previous post, EZH2 is hyperactive (=overexpressed) in many types of cancer, including breast and prostate cancers and, of course!, myeloma. I have therefore been looking for PubMed studies on non-toxic substances that can perhaps help us restrain the antics of this particularly mischievous Polycomb repressor gene.

A September 2009 study (see: http://tinyurl.com/382xp66) confirms that the Aberrant expression of EZH2 has been associated with metastasis and poor prognosis in cancer patients. It then adds that, in spite of EZH2’s involvement in oncogenesis and therapy failure, not much is known about chemotherapeutics and chemopreventive agents that can suppress its expression and activity. Here, we show that dietary omega-3 […] polyunsaturated fatty acids (PUFAs) can regulate the expression of EZH2 in breast cancer cells.

Oh wait, duuuh, I just quoted from the publicly-available abstract…my apologies! But I did so because it makes an important point: in spite of the evidence pointing to EZH2 as a prime target in cancer treatment, very little is known about its potential inhibitors…Well, we have seen that curcumin is one such inhibitor…and today’s study shows that dietary omega-3 PUFAs (=polyunsaturated fatty acids) are also able to block EZH2.

The full study (that I received from a very kind blog reader–thank you!) begins with a description of the Polycomb group proteins (PcGs) and the Polycomb repressive complexes (PRCs), which are formed when PcGs stick to one another…okay, skip skip skip…this stuff is giving me a headache…I need to forge ahead and select only the important bits…

Here is one: Chemopreventive agents such as dietary polyunsaturated fatty acids (PUFAs) are known to influence the development and progression of breast cancer and other cancers. Omega-3 PUFAs lower a woman’s risk of developing breast cancer, whereas omega-6 PUFAs are associated with a higher risk. However, the study informs us that even though the molecular targets of PUFAs are not very well understood, one of these targets happens to be EZH2…Well, that is cause for celebration, I’d say!

The researchers examined DHA and EPA, two of the best-known omega-3 PUFAs, as well as LA and AA, which are omega-6 PUFAs*. EZH2 became less active when breast cancer cells were treated with DHA and EPA, whereas it was not affected at all by AA and LA. The study describes a series of experiments, but the language used is very technical, so yes, I think I will go directly to the Discussion part…

Here we learn (…but are undoubtedly not surprised!) that omega-3 PUFAs have been the object of many cancer studies. Out of curiosity, I went to PubMed and found hundreds of studies, including this pancreatic cancer one: http://tinyurl.com/35syxy4. Apparently, omega-3s can mediate cancer development and progression through multiple mechanisms. For example, they have an impact on NF-kappaB. Good news for us, obviously.

Skipping on…Recent genetic studies suggest that PUFAs are involved in the regulation of a broad spectrum of genes involved in diverse biological functions such as nutrition, cell division, proliferation, apoptosis and metastasis. Omega-3s can inhibit angiogenesis (=another process that helps keep MM cells alive and healthy) and COX2 (yep, this one, too, is another big friend of myeloma cells). Well, the list goes on and on. Let’s leave it and get back to our point…

The point is that DHA and EPA, two omega-3 PUFAs,, can decrease the activity of (=down-regulate) EZH2, one of the blasted Polycomb repressor genes that is involved in the proliferation of myeloma cells.

In the researchers’ own words, our studies suggest that anti-oncogenic effect of omega-3 PUFAs, at least in part is mediated by the PcG targets of PUFAs such as EZH2. And, they add, to the best of our knowledge, this is the first study that links the regulation of expression of PcG proteins, in particular EZH2, to chemopreventive agents such as dietary fatty acids. Let’s hope that it is not the last!

Even though I have to admit that I didn’t understand a good part of this study (e.g., the bits about EZH2 posttranslational regulation and EZH2 ubiquitination and subsequent degradation by proteosome-dependent pathways…Aaagh!!!), this much is crystal clear: we should, indeed we MUST include omega-3 PUFAs in our diet. I take 2 grams of molecularly-distilled fish oil every day and, after reading this study, have found another excellent reason for sticking to my protocol…

*More information on the difference between omega-3 and omega-6 PUFAs: http://tinyurl.com/5e33wd and http://tinyurl.com/3ywwf8f (there is a ton of information out there on omega-3s and omega-6s…a quick search is all you need to do…).

August 2 2010 post. Thanks to a fabulous blog reader, I was able to read the entire text of a recently-published “Carcinogenesis” study on how EGCG, the main polyphenol in green tea, affects not one, not even two, but THREE (!) of the pesky Polycomb repressor genes that play central roles in myeloma. The study examines skin cancer cells, not myeloma ones, but the findings are certainly relevant for us, too. Here is the link to the abstract: http://tinyurl.com/32edo5d

We have already spent a lot of time on EZH2 (see previous posts), which is closely linked to the proliferation (etc.) of myeloma cells, so I won’t bother going over that one again.

But this is the first time we have dealt with Bmi-1 in connection with myeloma. All we need to do is take a look at the title of another study (abstract: http://tinyurl.com/32lsuf7): “The Polycomb Group Protein Bmi-1 Is Essential for the Growth of Multiple Myeloma Cells.” This part says it all: “…essential for the growth of myeloma cells…”

As for SUZ12, this Polyhooligan* (*this word does not exist, by the way…it just popped into my head as I was writing one of my Polycomb posts) is also super-active in multiple myeloma and, e.g., chronic myeloid leukemia. I became aware of its existence while going through the study that began my Polycomb quest: http://tinyurl.com/3y8errz This study mentions that SUZ12 is significantly correlated with the establishment and progression of MM. Eeek! 

So let’s have a look at the “Carcinogenesis” study. Today I don’t feel like quoting and commenting on huge portions of a study, so I will be amazingly brief…for a change.

In the abstract, you can read about EGCG’s anticancer activities…I wanted to highlight only that it inhibits some of myeloma’s best buddies—cyclin D1 and so on. Yes, you are right: the abstract is not easy to read (…you should see the full study!…Mamma mia!). In a nutshell, though, it tells us that EGCG reduces the survival of skin cancer cells by decreasing the mad activity of two Polyhooligans, Bmi-1 and EZH2. The third Polyhooligan, SUZ12, is discussed only in the full study (a bit of technical information: SUZ12 belongs to the PCR2 group).

Full study, now. It points out that EGCG has not (yet!) been studied in connection with the hyperactive Polycomb repressor genes. Other studies have shown that EGCG blocks the growth and indeed increases apoptosis in skin cancer cells (apoptosis is the process of programmed cell death). This study shows that expression of prosurvival PcG proteins is increased in skin cancer cells as compared with normal and that EGCG treatment of the cancer cells suppresses PcG protein expression and histone methylation leading to reduced cell survival. Basically, this means that skin cancer cells contain a whole bunch of these hyperactive Polyhooligans, which help them survive…but most of them begin dying as soon as EGCG enters the picture. Not bad, eh! 

Skipping a lot of details (a promise is a promise…!) and getting to the Conclusion: by reducing the hyperactivity of the three above-mentioned Polyhooligans, EGCG is able to have a huge impact on the survival of skin cancer cells. Gollywobbly!

August 29 2010 post. Yesterday, as I was going through my Science Daily newsletters, I found an article on the effect that EZH2 has on ovarian cancer cells (do you remember the Polyhooligans? Yep, EZH2 is one of those pesky Polycomb repressor genes that I began calling “Polyhooligans.” To brush up on your PcGs, look over on the right and scroll down to my Page titled “Myeloma and Polycomb repressor genes”).