Identification of microRNAs that stabilize p53 in HPV-positive cancer cells

Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p that has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs in addition to 375-3p were further assessed using a second cell-based p53 stability reporter system as well as in non-reporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. Importance Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high throughput live cell assay we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

whose expression is required for cancer initiation and maintenance. Among their 23 cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. 24 Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP 25 ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We 26 utilized a live cell high throughput screen to determine whether exogenous 27 microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive 28 cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of 29 p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 30 stabilization was 375-3p that has previously been reported to stabilize p53 in HeLa 31 cells, providing validation of the screen. The top 32 miRNAs in addition to 375-3p 32 were further assessed using a second cell-based p53 stability reporter system as 33 well as in non-reporter HeLa cells to examine their effects on endogenous p53 34 protein levels, resulting in the identification of 23 miRNAs whose transfection 35 increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to 36 decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We 37 further examined subsets of these miRNAs for their abilities to induce apoptosis 38 and determined whether it was p53-mediated. The introduction of specific miRNAs 39 revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, 40 some of the miRNAs described here have potential as therapeutics for treating HPV-41 positive cancers.

52
Cervical cancer is the second leading cause of cancer deaths among women 53 worldwide, with approximately 500,000 new cases diagnosed and 275,000 deaths each 54 year. Virtually all cases of cervical cancer are attributable to infection by HPVs. There are 55 over 200 different HPV types and a subset of these are linked to anogenital tract infections, 56 14 of which are referred to as hrHPV because they are associated with lesions that can 57 progress to cancer (1). These same hrHPVs are also associated with other genital tract 58 cancers (penile, vaginal, vulvar, and anal), as well as an increasing number of 59 oropharyngeal cancers. Indeed, approximately 5% of all cancers worldwide are caused by 60 hrHPVs (2). 61 Despite the success of the virus like particle (VLP)-based vaccine that has the 62 potential to prevent millions of new HPV-associated cancers, there is the need for effective 63 therapies to treat cancers that will develop in individuals who have not been vaccinated and those already infected with hrHPVs. In this regard, hrHPVs provide a number of 65 potential viral targets for specific HPV antiviral therapies, including the viral encoded 66 oncoproteins E6 and E7, the viral E1 helicase, and the viral E2 regulatory protein. 67 The hrHPVs associated with cervical cancer encode two oncoproteins, E6 and E7, 68 that are invariably expressed in HPV-positive cancers and target the important cellular 69 growth regulatory proteins p53 and pRb, respectively (1). These cancers are dependent on 70 the continued expression of E6 and E7 and silencing these oncoproteins results in 71 apoptosis and senescence (3, 4). The E6 proteins of the hrHPV types promote the ubiquitin-72 dependent degradation of the p53 protein (5) through its binding to the E3 ubiquitin ligase 73 E6AP (also known as UBE3A), which ubiquitylates p53 (6-8). In promoting the ubiquitin-74 dependent proteolysis of p53, E6 counters E7-mediated replication stress signals that 75 activate and stabilize p53 (9). Thus, inhibition of the E6/E6AP-mediated ubiquitylation of 76 p53 is pro-apoptotic and provides a validated target for HPV-positive cancers. For example, 77 direct targeting of E6 induces apoptosis in HPV-positive cancer cells (10). Although the 78 hrHPV E6 proteins have other cellular activities that have been described in the literature, 79 we have focused on the E6/E6AP pathway as a potential therapeutic target for HPV-80 positive cancers, as well as precancerous lesions. 81 MicroRNAs (miRNAs/miRs) are small non-coding RNAs (approximately 22 82 nucleotides in length) that significantly impact and regulate many essential biological 83 pathways. miRNAs negatively regulate gene expression either by translational repression 84 or cleavage of target mRNAs. Many miRNAs have been shown to play roles in cancer, 85 sometimes as oncogenes and other times as tumor suppressors, making them suitable as 86 therapeutic targets. miRNAs themselves can be used as cancer therapeutics as they may 87 affect one or more pathways essential for the replication and survival of specific cancer 88 cells. There have been a number of studies focused on HPV and miRNAs in recent years. 89 These studies have examined whether HPVs, like many other viruses, encode viral miRNAs 90 (11) and whether the HPV oncoproteins regulate the expression of cellular miRNAs (12). 91 Other studies have proposed miRNAs as potential biomarkers of hrHPV infections (13). 92 There have also been studies that examined the effects of miRNA expression in various 93 cancers including cervical cancers (For review see (14)). It was with this mindset that we 94 conducted a screen to identify miRNAs whose transfection into HPV-positive cancer cells 95 stabilize p53. 96 Here we present a high throughput screen using a cell-based platform to assay p53 97 stability in hrHPV E6-expressing HeLa cells that identified miRNAs whose transfection 98 increased p53 protein levels. Several of these miRNAs caused apoptosis and/or p21 99 induction when transfected into hrHPV-positive cells. The goal of this work was to find 100 miRNAs that could be used as therapeutic tools for the treatment of hrHPV-associated 101 diseases, regardless of whether these miRNAs are physiological regulators of p53 stability. 102 To our knowledge, this is the first study to systematically exam miRNAs for their abilities to 103 stabilize p53 in hrHPV-positive cancer cells. We also provide insights into the mechanisms 104 by which some of these miRNAs function in stabilizing p53 and promoting apoptosis. 105

106
A high throughput screen identified transfected miRNAs that 107 stabilize p53 in HeLa cells. 108 To identify miRNAs that stabilize p53 and induce apoptosis when transfected into 109 hrHPV-positive cancer cells, we performed a high throughput screen in HPV18-positive 110 HeLa cells harboring a reporter for p53 protein stability. The reporter was originally 111 designed by the Elledge lab for their "global protein stability system"(15, 16), and 112 expresses a bicistronic mRNA that encodes an EGFP-p53 fusion protein and the red 113 fluorescent protein DsRed as reference ( Figure 1A). The stability of the EGFP-p53 fusion 114 protein is determined by the less stable protein, in this case p53, with a half-life less than 115 30 minutes in HeLa cells due to E6/E6AP-mediated proteolysis (5, 17). To detect changes 116 in p53 stability, we monitored the EGFP/DsRed fluorescence ratio by laser scanning 117 cytometry. In our screen, for the fusion protein we used a p53 gene encoding the R273C 118 mutant, which is also efficiently targeted by E6/E6AP for ubiquitin-dependent proteolysis 119 (18). Because this mutant is defective in DNA binding (19, 20) and probably in the p53 120 mitochondrial pro-apoptotic pathways (21), it does not add an extra pro-apoptotic 121 stimulus to the eventual stabilization of the endogenous p53 during the course of the assay. 122 Furthermore, p53(R273C) can interfere with p53 transcriptionally driven apoptosis (22), 123 thus facilitating the acquisition of data for p53 stabilizers that might otherwise kill all cells. 124 The results of this screen, which assayed 885 miRNA mimics, are shown in Figure 1B and 125 Table S1. The top hit, 375-3p, has been previously reported to stabilize p53 in HPV-positive 126 cells (23) providing validation for this screen. Of note, since all the miRNAs used in this 127 study are human, we omitted the use of the prefix hsa-miR in the miRNAs names. We 128 selected for further testing the miRNAs that produced the next 32 highest EGFP/DsRed 129 fluorescence ratios (Table 1). 130 To confirm the results obtained in the screen, we tested the selected miRNAs in 131 HeLa cells containing a different bicistronic reporter vector that has been successfully used 132 in a small molecule screen to find p53 stabilizers in HeLa cells (24). In this vector, 133 p53(R273C) is fused to mRuby, a bright monomeric red fluorescent protein, and the 134 reference monomeric green fluorescent protein SGFP2 is fused to H2B (Figure 2A). For this 135 experiment, we determined the percentage of H2B-SGFP2 expressing cells that also 136 expressed mRuby-p53 (R273C) using flow cytometry. Two randomly selected miRNAs 137 (498-5p and 665), which were negative in the initial screen, and the miRNA Ctrl. 1 were 138 used as negative controls, along with 375-3p used as a positive control. As shown in Figure  139 2B, 25 of the 32 originally selected miRNAs were confirmed as significantly stabilizing 140 mRuby-p53 (R273C) in this reporter HeLa cell line. 141

202
Since the increase of p53 in HeLa cells transfected with the miRNAs identified in this 203 study negatively correlated to the levels of E6 ( Figure 4G), we hypothesized that the ability 204 of these miRNAs to stabilize p53 in HPV-positive cancer cells might be mediated, at least in 205 part, by a negative effect on E6 expression. This could be a consequence of destabilizing the 206 E6 protein or decreasing its expression, either by targeting the E6/E7 mRNAs for 207 degradation, preventing their translation, or decreasing viral transcription. We observed 208 that transfection of these miRNAs into HeLa cells, particularly members of the 302/519 209 family and 1287-5p, decreased both E6 and E7 protein levels similarly ( Figure 7A). This is 210 consistent with the idea that these miRNAs affect the expression of the viral E6/E7 mRNAs 211 since the hrHPV E6 and E7 genes are expressed from a single promoter located in the viral 212 LCR. This effect on the level of the E6/E7 mRNA in HeLa cells was confirmed by qRT-PCR 213 using primers that detect all HPV E6/E7 transcripts ( Figure 7B). Interestingly, all miRNAs 214 tested, including 375-3p and 148b-5p that increased p53 protein levels in the absence of 215 hrHPV proteins ( Figure 6F), also decreased the levels of the E6/E7 transcripts. Although 216 498-5p reduced the level of the E6/E7 mRNAs in HeLa cells, consistent with the reduced 217 level of E6 protein shown in Figures 3, 4 and 7A, we did not observe an increase in p53 218 protein levels (Figures 3 and 6). While 375-3p was as efficient in repressing the expression 219 of the E6/E7 mRNAs in HPV16 SiHa cells as in HPV18 HeLa cells, most of the tested 220 miRNAs were less efficient decreasing the levels of the E6/E7 transcripts in SiHa cells, even though they were efficiently transfected as determined by siGlo RISC-free transfection 222 control (data not shown). In addition to 375-3p, only 519b-3p and 1287-5p showed 223 comparable effects on the E6/E7 transcripts in both HPV-positive cell lines. Surprisingly, 224 148b-5p, which stabilized p53 in SiHa cells, produced an increase in the levels of the E6/E7 225 mRNAs but we could not confirm whether E6 protein levels were affected in this cell line 226 (e.g. by preventing translation) because we were unable to detect HPV16 E6 using any of 227 the commercially available anti-HPV16 E6 antibodies. 228 To determine whether these miRNAs were targeting the untranslated regions (UTR)  apoptosis is also p53-independent and consistent with the inability of this miRNA to 290 stabilize p53 when transfected into hrHPV-positive cell lines (Figures 3 and 6). Similarly, 291 the apoptotic effects of miRNAs 148b-5p and 374a-3p are also independent of p53, as co-292 transfection of these miRNAs with either the siRNA against p53 or its C911 version have 293 similar effects on the percentage of apoptotic cells. This indicates that, in these cases, the 294 effect observed when p53 is knocked down can be attributed to off-target effects of the 295 siRNA against p53. In contrast, the numbers of apoptotic cells observed with 519b-3p (as a 296 representative of the 302/519 family of miRNAs) and 1287-5p were reduced when co-297 transfected with the p53 siRNA but not with its C911 version, suggesting that the apoptotic 298 effect of these miRNAs is largely dependent on p53 in HeLa cells. Similar results were 299 observed when using PARP cleavage as a marker for apoptosis in western blot analysis (Fig  300   9B). 301

302
In this study we identified several miRNAs that increased p53 protein levels when 303 transfected in HeLa cells. Some of the miRNA hits from our high throughput screen (148b-304 5p, 302a-3p, 373-3p, and 374b-5p) have been previously reported to have decreased 305 expression in cervical cancer cells and tissues compared to the controls used in each of the 306 studies (26, 30-32). Another positive miRNA from the screen, 1287-5p, has been found to 307 have no change in its expression level in cervical cancer cells, but is sequestered away from 308 its targets by the circular RNA circSLC26A4, which is overexpressed in hrHPV-positive 309 cancer cells (33). Together these findings suggest that interfering with the functions of 310 some of the miRNAs identified in this work might contribute to the establishment of HPV 311 infections or/and progression of hrHPV-infected cells towards cancer. Consistent with that, 312 several of the miRNAs identified in our screen, namely 148b-5p, 302a-3p, 374b-5p, or 313 519b-3p, have been shown to slow cell growth and induce apoptosis in hrHPV-positive 314 cervical cancer cells (28, 31, 32, 34, 35). Taken together, these previous findings validate 315 this screening platform as a reliable tool to identify miRNAs that affect the cell growth and 316 survival of hrHPV-positive cells, as expected from miRNAs whose transfection stabilize 317

p53. 318
Since the ubiquitin ligase activity of E6AP/E6 is responsible for the efficient 319 proteolysis of p53 in hrHPV-positive cervical cancer cells, we determined whether 320 transfection of any of the miRNAs identified in this screen altered the levels of E6AP in 321 HeLa cells. Interestingly, only three of the 27 miRNAs analyzed in HeLa cells, 148b-5p, 322 374a-3p, and 374b-5p, led to a decrease in E6AP protein levels comparable to the decrease 323 produced by 375-3p. Although we did not determine whether they directly bind to the 324 E6AP mRNA, target prediction analysis using the miRDB database (www.mirdb.org) (36, 325 37) revealed that 148b-5p and 374b-5p each have three sites complementary to their seed 326 sequences within the 3'UTR of the E6AP mRNA. Remarkably, these four miRNAs (148b-5p, 327 374a-3p, 374b-5p, and 375-3p) also led to decreased E6 expression. This simultaneous 328 decrease of both E6AP and E6 may be required to stabilize p53 in HeLa cells, since a small 329 amount of E6AP is sufficient to efficiently promote p53 degradation in this cell line 330 (38). The need to suppress the very efficient E6/E6AP-mediated degradation of p53 to 331 increase the levels of this protein in hrHPV-positive cancer cells is exemplified by miRNAs 332 375-3p and 1287-5p. Both miRNAs stabilized p53 in absence of E6 when transfected into 333 U2OS cells, however, they also efficently decrease the levels of E6 when transfected into 334 HeLa and SiHa cells, suggesting that this is a necessary condition to stabilize p53 in these 335 cells. Moreover, in this study we haven't found any miRNA that stabilize p53 in hrHPV-336 cancer cells that do not significantly decrease the E6 protein level too. In contrast, 337 transfection of 498-5p, which also resulted in decreased levels of E6 in these cells, did not 338 stabilize p53 in HeLa cells, suggesting that the remaining E6 protein was sufficient to target 339 p53 for degradation. In line with this idea, in our study, miRNAs that failed to reduce E6 340 protein levels to approximately half of its original amount or beyond also failed to increase 341 p53 levels in HeLa cells, suggesting that there is a considerable excess of E6 in these cells 342 that must be overcome to stabilize p53. High levels of expression of E6 could therefore be a 343 limiting factor for the use of some miRNAs such as 498-5p as therapeutics, at least in some 344 hrHPV-associated cancers. 345 While several of the transfected miRNAs were found to induce apoptosis, the 346 mechanisms by which this occurred varied. The apoptotic induction in HeLa cells for three 347 of the miRNAs, 148b-5p, 374a-3p, and 375-3p, was independent of p53 stabilization. In 348 contrast, transfection of 1287-5p induced apoptosis in HeLa cells in a p53-dependent 349 manner. The miRNA 1287-5p was also found to efficiently induce apoptosis in U2OS cells 350 without a noticeable increase in p53 levels and to increase the number of apoptotic C33A 351 cells, which do not express wt p53, indicating that this miRNA promotes cell death 352 independent of p53 stabilization in these HPV-negative cells. Therefore, 1287-5p can 353 induce apoptosis via different pathways depending on the cell type. This is also exemplified 354 by Ctrl. 1 whose transfection stabilized p53 only in U2OS cells. The other miRNA that we points. However, we both found that 519b-3p transfection did not induce apoptosis in SiHa 362 cells, but led to an increase in p53 and p21 protein levels. It has been reported that p53 363 must accumulate to a threshold level to induce apoptosis (39). Thus, it is feasible that 364 under the experimental conditions used in our study 519b-3p did not increase p53 levels to 365 the threshold necessary to trigger apoptosis in SiHa cells. In addition, we observed that 366 transfection of all miRNAs belonging to the 302/519 family, with the exception of 519d-3p 367 in SiHa cells, increased p21 protein levels in hrHPV-positive cells indicating this is a family 368 characteristic rather than a feature of 519b-3p alone. 369 Throughout this study, 519d-3p has been less efficient than the other members of 370 the 302/519 family of miRNAs in promoting increased p53 and p21 protein levels in the 371 hrHPV-positive cell lines, as well as in repressing expression from the HPV16 and HPV18 372 LCRs, and inducing apoptosis in HeLa cells. In addition to a few base substitutions in the 3' 373 end sequences, a difference between 519d-3p and the other miRNAs of this family included 374 in this study is the addition of a C at its 5'end. This additional nucleotide produces a 375 displacement of the miRNA sequences, including its seed sequence, which is 5'-AAAGUG-3' 376 instead of 5'-AAGUGC-3', the most common seed sequence between the members of the 377 302/519 family. Since a seed sequence match is very important for miRNA target The utility of miRNAs as therapeutic tools will be improved by a more complete 394 understanding of how they function in the cellular networks in which they are embedded. 395 In this study, we identified several miRNAs that stabilize p53 in hrHPV-cancer cells and 396 induce apoptosis in HeLa cells in a p53-dependent or in p53-independent manner. Despite 397 the heterogenicity of the responses to different miRNAs, decreasing E6 protein levels 398 seems to be required to increase p53 protein levels in HeLa cells since all miRNAs found to 399 stabilize p53 also affected the expression of E6. Further research into the mechanisms 400 high throughput format. The Z'-factor (48) was calculated for each assay plate and was 432 consistently > 0.6, indicating assay robustness. siRNA buffer (1×) (Horizon #B-002000-UB-433 100) was aliquoted into wells, miRNA mimics were added so that the final concentration 434 was 40 nM/well, and DharmaFECT1/OptiMEM was dispensed into wells. While the 435 miRNA/lipid mix was allowed to complex, cells were trypsinized, counted, and 436 resuspended to reach a plating density of 600 cells/well. Cells were seeded on top of the 437 miRNA/lipid mixture, briefly centrifuged, and incubated for 72 hours, a time that we 438 determined to be optimal for p53 stabilization for small miRNAs. The cells were then 439 equilibrated to room temperature (~15 min) before analysis. Using TTP LabTech's Acumen   Dharmacon C-300615-07-0002 miRIDIAN microRNA Human hsa-miR-519a-3p -Mimic, 2 nmol Dharmacon C-300836-05-0002 miRIDIAN microRNA Human hsa-miR-1287-5p -Mimic, 2 nmol Dharmacon C-301341-00-0002 miRIDIAN microRNA Human hsa-miR-373-3p -Mimic, 2 nmol Dharmacon C-300680-03-0002 miRIDIAN microRNA Human hsa-miR-302a-3p -Mimic, 2 nmol Dharmacon C-300653-05-0002 miRIDIAN microRNA Human hsa-miR-302b-3p -Mimic, 2 nmol Dharmacon C-300669-05-0002 miRIDIAN microRNA Human hsa-miR-509-3p -Mimic, 2 nmol Dharmacon C-300849-07-0002 miRIDIAN microRNA Human hsa-miR-210-3p -Mimic, 2 nmol Dharmacon C-300565-03-0002 miRIDIAN microRNA Human hsa-miR-302c-3p -Mimic, 2 nmol Dharmacon C-300671-05-0002 miRIDIAN microRNA Human hsa-let-7a-2-3p -Mimic, 2 nmol Dharmacon C-301297-00-0002 miRIDIAN microRNA Human hsa-miR-224-5p -Mimic, 2 nmol Dharmacon C-300581-07-0002 miRIDIAN microRNA Human hsa-miR-302d-3p -Mimic, 2 nmol Dharmacon C-300672-05-0002 miRIDIAN microRNA Human hsa-miR-520e -Mimic, 2 nmol Dharmacon C-300772-03-0002 miRIDIAN microRNA Human hsa-miR-520a-3p -Mimic, 2 nmol Dharmacon C-300788-03-0002 miRIDIAN microRNA hsa-miR-20a-3p mimic, 2 nmol