Human papillomavirus type 8 oncoproteins E6 and E7 cooperate in downregulation of the cellular checkpoint kinase-1: HPV8 downregulates Chk1

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Abstract

Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus.

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Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage.

Jiri Bartek, Robin Hansen, Randi G. Syljuåsen … (2005)

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.

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Regulated Degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage

Caroline Park, Yousin Suh, Ana Maria Cuervo (2015)

Chaperone-mediated autophagy (CMA) is activate in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and aging. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

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Constitutive Autophagy and Nucleophagy during Epidermal Differentiation.

Olufolake A. Akinduro, Katherine Sully, Ankit Patel … (2016)

Epidermal keratinocytes migrate through the epidermis up to the granular layer where, on terminal differentiation, they progressively lose organelles and convert into anucleate cells or corneocytes. Our report explores the role of autophagy in ensuring epidermal function providing the first comprehensive profile of autophagy marker expression in developing epidermis. We show that autophagy is constitutively active in the epidermal granular layer where by electron microscopy we identified double-membrane autophagosomes. We demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of microtubule-associated protein light chain 3/lysosomal-associated membrane protein 2/p62 positive autolysosomes. These perinuclear vesicles displayed positivity for histone interacting protein, heterochromatin protein 1α, and localize in proximity with Lamin A and B1 accumulation, whereas in newborn mice and adult human skin, we report LC3 puncta coincident with misshaped nuclei within the granular layer. This process relies on autophagy integrity as confirmed by lack of nucleophagy in differentiating keratinocytes depleted from WD repeat domain phosphoinositide interacting 1 or Unc-51 like autophagy activating kinase 1. Final validation into a skin disease model showed that impaired autophagy contributes to the pathogenesis of psoriasis. Lack of LC3 expression in psoriatic skin lesions correlates with parakeratosis and deregulated expression or location of most of the autophagic markers. Our findings may have implications and improve treatment options for patients with epidermal barrier defects.