NR2E3 is a key component in p53 activation by regulating a long noncoding RNA DINO in acute liver injuries

2020

J Virol

Human papillomavirus 16 (HPV16) E7 has long been known to stabilize the tumor suppressor TP53. However, the molecular mechanism of TP53 stabilization by HPV16 E7 has remained obscure, and this stabilization can occur independently of the E2F-regulated MDM2 inhibitor p14ARF. Here, we report that the damage-induced noncoding (DINO) lncRNA (DINOL) is the “missing link” between HPV16 E7 and increased TP53 levels. DINO levels are decreased in cells where TP53 is inactivated, either by HPV16 E6, by expression of a dominant negative TP53 minigene, or by TP53 depletion. DINO levels are increased in HPV16 E7-expressing cells. HPV16 E7 causes increased DINO expression independently of RB1 degradation and E2F1 activation. Similar to what is seen with the adjacent CDKN1A locus, DINO expression is regulated by the histone demethylase KDM6A. DINO stabilizes TP53 in HPV16 E7-expressing cells, and as it is a TP53 transcriptional target, DINO levels further increase. As with expression of other oncogenes, such as adenovirus E1A or MYC, HPV16 E7-expressing cells are sensitized to cell death under conditions of metabolic stress, which in the case of E7 has been linked to TP53 activation. Consistent with earlier studies, we show that HPV16 E7-expressing keratinocytes are highly sensitive to metabolic stress induced by starvation or the antidiabetic drug metformin. Sensitivity of HPV16 E7-expressing cells to metabolic stress is rescued by DINO depletion. Moreover, DINO depletion decreases sensitivity to the DNA damage-inducing chemotherapy agent doxorubicin. This work identifies DINO as a critical mediator of TP53 stabilization and activation in HPV16 E7-expressing cells. IMPORTANCE Viral oncoproteins, including HPV16 E6 and E7, have been instrumental in elucidating the activities of cellular signaling networks, including those governed by the TP53 tumor suppressor. Our study demonstrates that the long noncoding RNA DINO is the long-sought missing link between HPV16 E7 and elevated TP53 levels. Importantly, the TP53-stabilizing DINO plays a critical role in the cell death response of HPV16 E7-expressing cells to metabolic stress or DNA damage.

Section: Discussionmentioning

confidence: 53%

KDM6A-Mediated Expression of the Long Noncoding RNA DINO Causes TP53 Tumor Suppressor Stabilization in Human Papillomavirus 16 E7-Expressing Cells

2020

J Virol

“…The initial trigger of HPV16 E7-mediated DINO induction that leads to TP53 stabilization and activation involves the H3K27 demethylase KDM6A (44), and HPV16 E7 is well known to cause double-strand DNA breaks (69,70). Similarly, an orphan nuclear receptor, nuclear receptor subfamily 2 group E member 3 (NR2E3), and the associated KDM1A (LSD1) H3K4 demethylase were shown to be necessary for efficient induction of DINO and TP53 activity in response to liver damage by N-acetyl-p-benzoquinone imine-mediated oxidative stress (71). It is noted that in addition to DNA damage, doxorubicin can also induce free radical release, thereby causing oxidative stress (72).…”

Section: Discussionmentioning

confidence: 99%

Expression of the Long Noncoding RNA DINO in Human Papillomavirus-Positive Cervical Cancer Cells Reactivates the Dormant TP53 Tumor Suppressor through ATM/CHK2 Signaling

2020

mBio

Tumor cells overcome the cytostatic and cytotoxic restraints of TP53 tumor suppressor signaling through a variety of mechanisms. High-risk human papillomavirus (HPV)-positive tumor cells retain wild-type TP53 because the HPV E6/UBE3A ubiquitin ligase complex targets TP53 for proteasomal degradation. While restoration of TP53 in tumor cells holds great promise for cancer therapy, attempts to functionally restore the dormant TP53 tumor suppressor in HPV-positive cancer cells by inhibiting the HPV E6/UBE3A ubiquitin ligase complex have not yet been successful. The damage-induced long noncoding RNA, DINO (DINOL), is a TP53 transcriptional target that has been reported to bind to and stabilize TP53, thereby amplifying TP53 signaling. We show that HPV-positive cervical carcinoma cells contain low levels of DINO because of HPV E6/UBE3A-mediated TP53 degradation. Acute DINO expression overrides HPV16 E6/UBE3A-mediated TP53 degradation, causing TP53 stabilization and increased expression of TP53 transcriptional target genes. This causes a marked sensitization to chemotherapy agents and renders cells vulnerable to metabolic stress. Acute DINO expression in HPV-positive cervical cancer cells induces hallmarks of DNA damage response signaling, and TP53 activation involves ATM/CHK2 signaling. DINO upregulation in response to DNA damage is independent of ATM/CHK2 and can occur in cancer cells that express mutant TP53. IMPORTANCE Functional restoration of the TP53 tumor suppressor holds great promise for anticancer therapy. Current strategies are focused on modulating TP53 regulatory proteins. Long noncoding RNAs (lncRNAs) have emerged as important regulators of TP53 as well as modulators of downstream tumor-suppressive transcriptional responses. Unlike many other cancer types, human papillomavirus (HPV)-positive cancer cells retain wild-type TP53 that is rendered dysfunctional by the viral E6 protein. We show that acute expression of the damage-induced long noncoding RNA, DINO, a known TP53 transcriptional target and functional modulator, causes TP53 reactivation in HPV-positive cervical cancer cells. This causes increased vulnerability to standard chemotherapeutics as well as biguanide compounds that cause metabolic stress. Hence, strategies that target DINO may be useful for restoring TP53 tumor suppressor activity in HPV-positive cancers and other tumor types that retain wild-type TP53.

“…The initial trigger of HPV16 E7 mediated DINO induction that leads to TP53 stabilization and activation involves the H3K27 demethylase KDM6A (44) and HPV16 E7 is well known to cause double strand DNA breaks (69,70). Similarly, an orphan nuclear receptor, Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3) and the associated KDM1A (LSD1) H3K4 demethylase were shown to be necessary for efficient induction of DINO and TP53 activity in response to liver damage by N-acetyl-p-benzoquinone imine mediated oxidative stress (71). It is noted that in addition to DNA damage, doxorubicin can also induce free radical release thereby causing oxidative stress (72).…”

Section: Discussionmentioning

confidence: 99%

Expression of the long noncoding RNA DINO in HPV positive cervical cancer cells reactivates the dormant TP53 tumor suppressor through ATM/CHK2 signaling

2020

Preprint

31Tumor cells overcome the cytostatic and cytotoxic restraints of TP53 tumor suppressor 32 signaling through a variety of mechanisms. High-risk human papillomavirus (HPV) positive 33 tumor cells retain wild type TP53 because the HPV E6/UBE3A ubiquitin ligase complex targets 34 TP53 for proteasomal degradation. While restoration of TP53 in tumor cells holds great promise 35 for cancer therapy, attempts to functionally restore the dormant TP53 tumor suppressor in HPV 36 positive cancer cells by inhibiting the HPV E6/UBE3A ubiquitin ligase complex have not yet 37 been successful. The Damage Induced long noncoding RNA, DINO, (DINOL) is a TP53 38 transcriptional target that has been reported to bind to and stabilize TP53, thereby amplifying 39 TP53 signaling. We show that HPV positive cervical carcinoma cells contain low levels of 40 DINO because of HPV E6/UBE3A mediated TP53 degradation. Acute DINO expression 41 overrides HPV16 E6/UBE3A mediated TP53 degradation, causing TP53 stabilization and 42 increased expression of TP53 transcriptional target genes. This causes a marked sensitization to 43 chemotherapy agents and renders cells vulnerable to metabolic stress. Acute DINO expression in 44 HPV positive cervical cancer cells induces hallmarks of DNA damage response signaling and 45 TP53 activation involves ATM/CHK2 signaling. DINO upregulation in response to DNA 46 damage is independent of ATM/CHK2 and can occur in cancer cells that express mutant TP53. 47 IMPORTANCE 49 Functional restoration of the TP53 tumor suppressor holds great promise for anti-cancer therapy. 50 Current strategies are focused on modulating TP53 regulatory proteins. Long noncoding RNAs 51 (lncRNAs) have emerged as important regulators of TP53 as well as modulators of downstream 52 tumor suppressive transcriptional responses. Unlike many other cancer types, human 53 papillomavirus (HPV) positive cancer cells retain wild type TP53 that is rendered dysfunctional 54 by the viral E6 protein. We show that acute expression of the Damage Induced long Noncoding 55 RNA, DINO, a known TP53 transcriptional target and functional modulator, causes TP53 56 reactivation in HPV positive cervical cancer cells. This causes increased vulnerability to standard 57 chemotherapeutics as well as biguanide compounds that cause metabolic stress. Hence, strategies 58 that target DINO may be useful for restoring TP53 tumor suppressor activity in HPV positive 59 cancers and other tumor types that retain wild type TP53. 60 61 62 63 64 65 KEYWORDS 66 Human papillomaviruses, cervical cancer, lncRNA, DINO, DINOL, TP53, UBE3A, E6-AP, 67 DNA damage, cellular transformation, chemoresistance, metabolism 68 69 RESULTS 126 127 Low DINO levels in HPV-positive cervical cancer cells are a result of HPV E6-mediated 128 TP53 degradation 129We previously reported that DINO expression correlated with TP53 levels in HPV E6 and/or E7 130 expressing human foreskin keratinocytes (HFKs) (44). Given that HPV positive cervical cancer 131 lines express E6 and E7 and retain wild-type TP53, we hypothe...