Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis

Abstract

The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.

Retroviruses of the Human Virobiota: The Recycling of Viral Genes and the Resulting Advantages for Human Hosts During Evolution

“…However, some HERVs are also compatible with parasitic relationship. In fact, various studies have highlighted an association between the presence of HERVs, probably those that have integrated into human genome more recently, and some rare and incurable diseases, in particular autoimmune, neurodegenerative, and cancer diseases (Antony et al, 2004;Tramontano, 2017, 2018a;Ibba et al, 2018). These findings are highly relevant because, if a clear evidence emerges that HERVs are explicitly involved in the onset of these pathologies, this would open up new treatment prospects, as they would no-longer be considered incurable diseases, but as pathologies involving one or more retroviruses against which antiretroviral therapies could be used or developed.…”

Human Endogenous Retrovirus K in Cancer: A Potential Biomarker and Immunotherapeutic Target

“…In vivo, HERV-K env RNA knockdown led to reduced metastasis [ 83 ]. Finally, the HERV-K Env protein is able to affect cellular networks and tumor-associated gene expression that play key roles in carcinogenesis (EGFR, c-Myc, TGFB1, NF-κB, p53, p-ERK, p-RSK, p-AKT and Ras) [ 76 , 83 , 84 ]. In particular, the HERV-K env gene can also produce Np9 or Rec proteins through alternative splicing from the env transcript.…”

Expression of HERV Genes as Possible Biomarker and Target in Neurodegenerative Diseases

“…The expression of HERV-K, especially of the HERV-K(HML-2) subgroup, has been associated with many cancers such as prostate cancer, melanoma, teratocarcinoma, ovarian, and germ cell tumours, as reviewed in [53]. Three mechanisms potentially involved in HERV-K oncogenesis have been described: i) the LTRs, which may enhance transcription of host genes; ii) HERV-K-encoded proteins, such as Np9 (a pivotal switch of several signaling pathways), and Rec (de-repressor of oncogenic transcription factors), both splicing products of the env gene; iii) the cytoplasmic tail of HERV-K(HML-2) Env, which, unique among the retroviral Env proteins tested, is oncogenic per se , is a strong inducer of several transcription factors associated with cellular transformation, as reviewed in [55]. This was confirmed by our finding that the disruption of the HERV-K(HML-2) env gene causes the downregulation of EGF-R and NF-κB, pivotal signaling pathways, central to cancer and immune responses [55].…”

“…To give insights on the HERV-K effects, Ibba and colleagues disrupted the HERV-K(HML-2) env gene by CRISPR/SaCas9 endonuclease technology, in cultured cells normally expressing HERV-Kenv mRNA and proteins [55]. When the HERV-K env gene was disrupted, there was a down-modulation of important regulators of cell expression and proliferation, involved in signaling, RNA-binding and alternative splicing, as EGF-R, NF-κB, SF2/ASF and TDP-43.…”