The genetic landscape of cancers is incredibly complex. Tumors lose and gain entire chromosomes, single base pairs are commonly mutated in important genes, and regions within chromosomes are rearranged, amplified, or deleted. The vast numbers of these genetic alterations make it difficult to determine the most important ones – scientists need to know which changes have no consequence and which ones initiate or facilitate tumor growth. Some genes like p53 are commonly altered across many cancer types, but many are unique to particular tumor types. At Fred Hutch, Dr. David MacPherson uses mouse models to identify the specific cancer pathways that cause the development of small cell lung cancer and retinoblastoma. In both of these malignancies it is common for the retinoblastoma tumor suppressor protein (pRB) to be mutated or deleted; however, within these tumor types different pathways combine with pRB loss resulting in unique physiologies. Dr. Nan Wu, a postdoctoral researcher in the MacPherson Lab, has recently characterized how MYCN amplification combines with pRB loss to drive retinoblastoma tumors. The findings published in the Journal of Clinical Investigation suggest MYCN is a useful therapeutic target for some retinoblastomas, but that tumor reemergence could be common.
MYCN is one of three MYC family members that drive transcription and are strongly linked to cancer development when their expression is increased. In the case of retinoblastoma, MYCN amplification (producing many copies of the gene) is commonly observed; however it was unknown if this event had any relevance on tumor biology. To test this, Dr. Wu and her colleagues generated genetic mouse models to determine how three genes combine to drive retinoblastoma. This was achieved by deleting two related proteins, pRB and p107, specifically in the retina and/or inserting an inducible form of an extra copy of the MYCN gene. For this study, mice were observed for about one year and no tumors were found in mice lacking pRB alone, while tumors were observed in 50% of mice ~230 days after deletion of both pRB and p107. When pRB was deleted and MYCN overexpressed, 100% of mice developed tumors within 150 days. Additionally deleting p107 in mice lacking pRb and overexpressing MYCN further decreased tumor latency. These results clearly demonstrated that amplified MYCN contributes to the initiation/growth of retinoblastoma in vivo.
The next step of this study was to characterize how pRB and MYCN work together to form these aggressive retinoblastoma tumors. In many tumors loss of pRB causes rapid cell cycling evidenced by more cells duplicating their DNA or more cells entering mitosis. This was the case in tumors from mice lacking pRB and was further increased when MYCN was also overexpressed. The loss of pRB activates the E2F family of transcription factors that transcribe a specific set of genes, yet MYCN is also a transcription factor with its own unique transcription targets. A likely hypothesis is that these two transcriptional activities work independently to increase tumor formation. However, when Wu and colleagues compared the RNA profiles of pRB deletion mice to pRB deletion with MYCN expression they found MYCN expression further increased the RNA levels of E2F targets. Thus while MYCN has unique targets it also exacerbated the effects of pRB loss. This cross-talk was particularly interesting to the researchers, “In the rare subset of human retinoblastomas that exhibit MYCN amplification in the context of wild-type RB, we suspect that evasion of pRB and inhibitory effects of pRB on E2F transcription is important. MYCN overexpression can drive E2F transcriptional programs in the retina. One of our aims in this project was to model MYCN-amplified, RB wild-type retinoblastoma to investigate this question. However, with the level of MYCN overexpression achieved in our genetic system, MYCN overexpression was not sufficient to drive retinoblastoma on its own; cooperation with RB deletion was still needed” said Dr. MacPherson.
In other cancers inhibiting the pathway that drives tumor formation has yielded successful therapies. In these instances the tumor become ‘addicted’ or dependent on the activated protein for survival. The result of inhibiting MYCN in this model was easily determined since over-expression of MYCN relied on doxycycline (dox) and removing the dox shuts off MYCN similar to a chemical inhibitor. Cells were isolated from the tumors of transgenic mice maintaining dox in the growth medium. Once dox was removed the cells stopped dividing, their chromatin had increased levels of modifications associated with transcriptional silencing, and they expressed markers of senescence. These findings were somewhat consistent in vivo; mice were fed dox-containing food until a visible tumor formed, then dox was removed. In 66/71 mice tumors actually reduced in size until they were no longer visible, on average this took 27 days. While tumor volumes were reduced, unlike in culture, the tumor cells did not express any senescence markers.
Tumors regressed in 66 mice after MYCN was inhibited, but 56 of these mice experience tumor recurrence. In some way tumors were growing without the overexpression of MYCN. When the genomes of the recurrent tumors were analyzed for deletions or amplifications, at least two mechanisms became clear. In this model, the dox-inducible MYCN was an extra copy on top of the two already coded in the mouse genome, and in one of these recurrent tumors the original MYCN gene was amplified, achieving the same effect. A more common amplification was a specific MYCN transcriptional target, a microRNA, miR-17-92. This result is connected to previous studies showing that miR-17-92 expression works with pRB loss to drive retinoblastoma.
This work validated MYCN amplification as an important genetic alteration in retinoblastoma that contributes to tumor proliferation. MYCN may also be an important therapeutic target in these cancers; however the miR-17-92 recurrence suggests that a combinatorial approach may be more appropriate.
Funding for this research was provided by the National Cancer Institute, the American Cancer Society, and the Elmer and Sylvia Sramek Foundation.
Wu N, Jia D, Bates B, Basom R, Eberhart CG, MacPherson D. 2017. A mouse model of MYCN-driven retinoblastoma reveals MYCN-independent tumor reemergence. Journal of Clinical Investigation, 127(3), 888-898. PMCID: PMC5330763.