Resistance of ER-positive breast cancer to tamoxifen therapy may be driven by APOBEC3B

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Published: 10 Dec 2015
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Dr Reuben Harris - Howard Hughes Medical Institute, Minneapolis, USA

Dr Harris presents, at a press conference at SABCS 2015 discussing the latest data on APOBEC3B. 

The data shows responses to tamoxifen were significantly prolonged by reducing levels of the enzyme APOBEC3B in preclinical models of oestrogen receptor–positive breast cancer, additionally there were significantly shortened by increasing levels of APOBEC3B, suggesting that APOBEC3B drives resistance to tamoxifen.

Read the news article here 

I’m going to talk to you about APOBEC. APOBEC is not one single protein, it’s a family of proteins; the key enzyme that we and others have described as mutating the cancer genome and contributing a lot to the overall genetic diversity in breast cancer is called APOBEC3B. The hallmark of this family member and others in the family is they catalyse the simple chemical reaction of converting cytosine bases to uracils and that’s a premutagenic lesion.

What we’re really interested in testing is the hypothesis that tumours are in fact achieving a mutator state and that APOBEC3B is a part of this elevated mutation state. So in the middle there depicted is a simple schematic essentially of a normal cell converting to a mutator state and there’s some tumour initiating event that we’re starting to learn more about, and there’s some references there for that, that lead to higher levels of this enzyme called APOBEC3B which leads to increased mutagenesis and everything associated with that, so increased tumour heterogeneity and any of the other mutation driving events including drug resistance. So how do we test this hypothesis? How is APOBEC3B making an impact on tumours?

To get a handle on this we collaborated with a Dutch group led by Jon Martens at Erasmus University and we took a cohort of 633 ER positive breast cancer, so this is a retrospective study, a cohort of 633, and we quantified APOBEC3B expression using assays that we have developed in my lab that are highly specific to this one family member and then split the cohort into groups with high levels and low levels of this particular enzyme.

These results are shown here and these data are published but they’re a segue into the actual results where one can see that over time for both disease free survival and overall survival there’s a significant difference between those that have high and low levels of this particular DNA mutating enzyme. This leads into the present work, which is unpublished and will be presented later today, where we wanted to look further into that to ask whether APOBEC3B is correlating with adverse outcomes including recurrence and resistance to tamoxifen therapy. This is also a retrospective study where upon diagnosis these tumours were surgically resected and then upon recurrence with knowledge that they were ER positive they were given tamoxifen monotherapy and then outcomes were monitored over time. This is a cohort of 285 that we’ve separated into quarters so that one can see the very dramatic effect of those with the highest levels of APOBEC3B faired much more poorly than those with the lowest levels and with intermediate levels tracking between those. This is a highly significant result with median APOBEC3B high progression free survival being about 7.5 months and those whose primary tumours, and I want to emphasise that the primary tumours is where we measured the APOBEC3B levels, had a much longer progression free survival.

So these are correlative studies that really suggest what many of the cellular experiments and genetic experiments in my lab and others have indicated that this would be an adverse effector in ER positive breast cancer but we wanted to test this hypothesis with a cause and effect experiment. So that’s shown here where we’ve been able to use a xenograft approach to take an oestrogen receptor breast cancer cell line, essentially graft it into mice at the time point shown there. Prior to engraftment actually we engineered the cells to have high and low APOBEC3B expression levels using shRNA technology and so we have those two arms. After those cell masses become small tumours at the time point about 15 weeks then we administer either tamoxifen monotherapy or the control arm and then follow these tumours over time.

So this is a lab model for tamoxifen treatment or hormone treatment of ER positive breast cancer. So, importantly, when we do this genetic engineering trick these are highly specific shRNA constructs that again, like the qPCR assays, do not effect, are very specific to APOBEC3B and have no off-targets with the other APOBEC family members. There’s no difference in the rates of cellular growth in cell culture in plastic. The doubling times are the same whether we’ve engineered them with the knockdown construct or the control. Just for your sake, these form relatively large tumour masses over time in the animals. Importantly, the durability of our knockdown lasts through the entire duration of the experiment which is a long-term, nearly a year long, experiment.

So here are the key data with the cause and effect relationship. In the absence of therapy, so the red and the blue lines depict growth of these cells in the animals over time, so just with estradiol provided in the drinking water, no tamoxifen therapy, they grow very rapidly into those large tumours I just showed you. In the presence of tamoxifen therapy applied at day 100 of age of these animals, one can see that those that had the normal levels of APOBEC3B in their cell line, those cell masses developed resistance to the tamoxifen therapy over time and by the end of the year-long experiment most of these animals had tumours that resisted drug therapy. But if we had knocked down this one single DNA mutating enzyme, one can see that the therapeutic durability is much more effective. So most of those animals did not develop resistance to the tamoxifen monotherapy and lived to the natural endpoint of their natural life expectancy.

So we wanted to go a little bit further and ask so if taking it down slows down essentially drug resistance if adding more of this enzyme speeds up drug resistance. So we can do that in this model system as well by adding either APOBEC3B to the cells, called MCF7L cells, or a catalytic mutant that E255Q shown there, so the red and the purple histogram graphs. Again the doubling time between these lines isn’t any different. This is the result, I’m sorry if you can’t quite see this but it’s on your handouts, this is essentially what I just showed you, that if we overexpress the catalytic mutant we don’t change the experimental result at all in that in the absence of therapy these cells grow into large tumour masses quickly; in the presence of therapy resistance develops relatively rapidly. So remember there are relatively high levels of endogenous APOBEC3B still in the system. But if we overexpress the wildtype enzyme that does have DNA deaminase activity one can see that the effect of tamoxifen is almost zero. So now instead of having durable therapy we have almost immediate resistance to tamoxifen.

So we can slow it down and we can speed it up and all of this is consistent with the clinical data indicating that those primary breast tumours that have high levels of this enzyme result in poorer outcomes than those tumours that have low levels. Again, this is an enzyme that’s a DNA cytosine deaminase that’s overexpressed in lots of breast cancers, we showed this previously. Some of you may be familiar with these mutation signatures, so it’s responsible for the second largest mutation signature observed in breast cancer. I just mentioned the clinical results and an important point to make is that unlike many of these things that are driving tumours at the genetic or molecular level, this is a gain of function enzyme and it’s not a loss of function, so taking away DNA repair or process like BRCA for instance. So an analogy I like to make to really drive home the message is that, relative to melanoma, so long ago they appreciated that UV rays were harmful and that they caused specific lesions in DNA and because medical companies were innovative in developing sunscreens that became better and better over time and people are a little bit… more and more protective clothing and that led to improved health. Now we’re in a similar state with APOBEC3B in that we’ve identified the mutational source, we’re beginning to appreciate the impact that it has on some types of cancers and we can think about innovative ways of stopping this enzyme and improving outcomes.