Paper Review: Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma

Hello everyone.

It has been bloody long since I last blogged because I have been battling illness and getting stuck into research at the same time. I’m going to review a paper I did some work towards in this post. I have written about DNA methylation in the past and the research groups I worked with/continue to work with were focusing on the various genetic and epigenetic alterations that characterise  head and neck squamous cell carcinoma (HNSCC), which is the sixth most common type of cancer.

HNSCC can be thought of as two distinct cancers with vastly different prognosis and aetiologies; a vast majority are caused by heavy smoking overlapping with heavy drinking and an increasing proportion is caused by HPV infection, transmissible through oral sex. (HPV, by the way, is the same virus that drives cervical cancer). HPV infection per se is insufficient to cause the cancers associated with it – there have to be additional genetic and epigenetic modifications on top. HPV positive HNSCC has excellent survival relative to HPV negative HNSCC, by the way.

In this study, we obtained clinical samples of both HPV positive and HPV negative HNSCC – some were fresh frozen upon surgical resection/biopsy, a lot were FFPE samples (Formalin fixed,paraffin embedded) and we also profiled cell lines using Illumina 450k methylation arrays, which give a read-out of methylation at 483,000 CpG sites (A cytosine followed by a guanine) across the human genome for less than a large pizza per sample.

The FFPE samples were used as a training set and the Fresh frozen samples and cell lines were used as a validation set. We found quite a few interesting things…

[1] HPV positive HNSCC exhibits a greater degree of DNA methylation (Hypermethylation) than HPV negative HNSCC, especially in genes that are known to be silenced by PRC2 complexes in stem cells. PRC2 complexes consist of multiple proteins that co-operate to produce the H3k27 histone mark. We find the same genes being silenced by DNA methylation instead in HPV positive HNSCC. This is also strongly associated with differences in expression; More the methylation, less the expression, as it should be.

HPV positive HNSCC exhibits hypermethylation relative to HPV negative HNSCC. Blue represents high methylation and yellow represents low methylation.

HPV positive HNSCC exhibits hypermethylation relative to HPV negative HNSCC. Blue represents high methylation and yellow represents low methylation.

[2] A subset of HPV positive HNSCC showed very high degrees of methylation – which is called a CpG Island Methylator Phenotype, and was associated with significantly worse survival.

CIMP phenotype (Cluster 1a) is associated with very high methylation, HPV positivity and significantly worse survival compared to HPV positive tumours with comparatively less methylation (Cluster 1b) as shown in the Kaplan Meier curve at the right.

CIMP phenotype (Cluster 1a) is associated with very high methylation, HPV positivity and significantly worse survival compared to HPV positive tumours with comparatively less methylation (Cluster 1b) as shown in the Kaplan Meier curve at the right.

[3] If you put the viral oncogenes E6 and E7 into a cell line that was derived from HPV negative HNSCC, you tend to see that E6 induces hypermethylation. This wouldn’t be surprising because p53, which is blocked by E6, is known to regulate DNMT1, a DNA methyltransferase that is involved in the maintenance of methylation.

[4] If you use probes on the array that are significantly different between HPV positive HNSCC and HPV negative HNSCC, and compare them to publicly available data for cervical and lung cancer by a process called multidimensional scaling, you find that HPV negative HNSCC is closely related to lung cancer while HPV positive HNSCC is closer to cervical cancer, suggesting that HPV modulates the methylation patterns that make cervical cancer closer to HNSCC of this type.

Multidimensional scaling shows HPV negative HNSCC (HPV0) to be more closely related to lung cancer and HPV positive HNSCC to be similar to cervical cancer.

Multidimensional scaling shows HPV negative HNSCC (HPV0) to be more closely related to lung cancer and HPV positive HNSCC to be similar to cervical cancer.

[5] The relationship between methylation and expression is valid and as predicted even in a panel of HNSCC cell lines, as I demonstrated using qPCR, where we get RNA, make DNA, and then do PCR to find out how many cycles it takes to get past a particular threshold of fluorescence.

Genes that are significantly hypermethylated in HPV positive HNSCC are relatively overexpressed in HPV negative HNSCC as expected (The relationship between most methylation and expression is inverse)

Genes that are significantly hypermethylated in HPV positive HNSCC are relatively overexpressed in HPV negative HNSCC as expected (The relationship between most methylation and expression is inverse)

[6] We found that DNMT1 and DNMT3a , which are enzymes involved in maintaining and establishing DNA methylation, are expressed more in HPV positive HNSCC cell lines relative to HPV negative HNSCC cell lines as a group.

DNMT1 and DNMT3a are significantly overexpressed in a panel of HPV positive HNSCC cell lines vs HPV negative HNSCC cell lines.

DNMT1 and DNMT3a are significantly overexpressed in a panel of HPV positive HNSCC cell lines vs HPV negative HNSCC cell lines.

So basically, we started off with two subsets of a type of cancer, identified that the methylation patterns between them are different, that this has functional ramifications and clinical implications. It would be very interesting if someone ended up looking at hitting methylation in HPV positive cancers with anti-methylation drugs to see if that high level of methylation is just an artefact of how HPV positive HNSCC develops or whether there are therapeutic opportunities to be had.

Journal Reference 
Matthias Lechner, Tim Fenton, James West, Gareth Wilson, Andrew Feber, Stephen Henderson, Christina Thirlwell,Harpreet K Dibra, Amrita Jay, Lee Butcher, Ankur R Chakravarthy, Fiona Gratrix, Nirali Patel, Francis Vaz, Paul O’Flynn, Nicholas Kalavrezos, Andrew E Teschendorff, Chris Boshoff and Stephan Beck, Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma, Genome Medicine 2013, 5:15 doi:10.1186/gm419
URL – http://genomemedicine.com/content/5/2/15

Cheers,
Exploreable.

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