Induction of Dormancy in Hypoxic Human Papillomavirus-Positive Cancer Cells

Induction of dormancy in hypoxic human papillomavirus-positive cancer cells

Karin Hoppe-Seylera, Felicitas Bosslera, Claudia Lohreya, Julia Bulkeschera, Frank Röslb, Lars Jansenc, Arnulf Mayerd, Peter Vaupeld, Matthias Dürstc, and Felix Hoppe-Seylera,1

aMolecular Therapy of Virus-Associated Cancers (F065), German Cancer Research Center, D-69120 Heidelberg, Germany; bViral Transformation Mechanisms (F030), German Cancer Research Center, D-69120 Heidelberg, Germany; cDepartment of Gynecology, Jena University Hospital, D-07743 Jena, Germany; and dDepartment of Radiooncology and Radiotherapy, Mainz University Medical Center, D-55131 Mainz, Germany

Edited by Karl Münger, Tufts University School of Medicine, Boston, MA, and accepted by Editorial Board Member Peter K. Vogt December 19, 2016 (received for review September 21, 2016) Oncogenic human papillomaviruses (HPVs) are closely linked to major (17), is considered to play a major role in tumor development and human malignancies, including cervical and head and neck cancers. It progression. Clinically, hypoxia can increase the resistance to is widely assumed that HPV-positive cancer cells are under selection chemotherapy and radiotherapy and is a negative prognostic pressure to continuously express the viral E6/E7 oncogenes, that their marker for many cancers, including HPV-positive tumors (15, 16, intracellular p53 levels are reconstituted on E6/E7 repression, and that 18–20). Notably, although O2 availability is known to affect tumor E6/E7 inhibition phenotypically results in cellular senescence. Here we cell biology (14–16), most functional studies of the HPV E6/E7 show that hypoxic conditions, as are often found in subregions of oncogenes in cervical cancer cells have been performed under cervical and head and neck cancers, enable HPV-positive cancer cells to standard cell culture conditions at 21% O2. In contrast, cervical escape from these regulatory principles: E6/E7 is efficiently repressed, cancers often exhibit strongly reduced O2 content, with a heter- yet, p53 levels do not increase. Moreover, E6/E7 repression under ogenous distribution of more- and less-oxygenated regions and a hypoxia does not result in cellular senescence, owing to hypoxia- median O2 concentration of 1.2% (16, 21). associated impaired mechanistic target of rapamycin (mTOR) signaling These considerations raise the question of whether our current via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest concepts about the interactions of the viral oncogenes with the host is induced that can be overcome by reoxygenation. Impairment of cell are mirrored under hypoxic conditions. In the present work, we mTOR signaling also interfered with the senescence response of hyp- found that hypoxic HPV-positive cancer cells strongly down-regulate oxic HPV-positive cancer cells toward prosenescent chemotherapy in E6/E7 expression, but this is not linked to a reconstitution of p53. vitro. Collectively, these findings indicate that hypoxic HPV-positive Notably, and in sharp contrast to their phenotype under normoxia, cancer cells can induce a reversible state of dormancy, with decreased we found that hypoxic HPV-positive cancer cells do not senesce viral antigen synthesis and increased therapeutic resistance, and may despite efficient E6/E7 repression. Instead, the cells switch to a serve as reservoirs for tumor recurrence on reoxygenation. dormant state, characterized by E6/E7 down-regulation and a reversible growth arrest. On reoxygenation, the dormant cells re- human papillomavirus | tumor virus | cervical cancer | hypoxia | mTOR store E6/E7 expression and resume proliferation. Mechanistically, we found that senescence induction on E6/E7 repression under ncogenic human papilloma viruses (HPVs) are some of the normoxia is critically dependent on intact mechanistic target of Omost important known cancer risk factors and are closely rapamycin (mTOR) signaling. Hypoxic HPV-positive cancer cells es- linked to the development of every 20th human cancer worldwide, cape from this regulation owing to the concomitant impairment of the including prevalent cancers in the oropharynx and anogenital re- mTOR pathway via the inhibitory REDD1/TSC2 axis. These results gion (1, 2). Best characterized is their causative role for cervical provide surprising insight into the functional cross-talk between the cancer, which alone accounts for more than 500,000 new cancer cases and more than 250,000 cancer deaths per year worldwide (3). Significance Cervical cancer cells virtually always contain the DNA of high-risk HPV types, such as HPV16 and HPV18. Maintenance of the Human papillomaviruses (HPVs) are major human carcinogens. It malignant phenotype of HPV-positive cancer cells is considered to is widely assumed that HPV-positive tumor cells must sustain viral require sustained expression of the viral E6/E7 oncogenes (1, 2). E6/E7 oncogene expression to continuously block the tumor- Inhibition of E6/E7 expression leads to the rapid induction of suppressive senescence response of the host cell. Consequently, – cellular senescence (4 6), a central tumorsuppressive pathway, E6/E7 are considered attractive therapeutic targets for immuno- resulting in an irreversible growth arrest (7). This indicates that the therapy or for functional inhibition. Here we show that hypoxic viral oncogenes maintain the growth of HPV-positive cancer cells conditions, as often found in HPV-positive cancers, allow the cells by blocking cellular senescence. However, their potential to induce to induce a dormant state in which E6/E7 is down-regulated but senescence on E6/E7 inhibition also shows that this pathway is not induction of senescence is avoided. Instead, a reversible growth irreversibly destroyed in HPV-positive cancer cells. arrest is induced that can be overcome by reoxygenation. As a These considerations are not only fundamental for our mecha- consequence, hypoxic HPV-positive cancer cells are protected nistic concepts of HPV-linked cell transformation, but also have against chemotherapy as well as against virus-specific therapeutic important therapeutic implications. The development of specific approaches, and may serve as reservoirs for cancer recurrence E6/E7 inhibitors could provide a rational strategy for targeting on reoxygenation. HPV-positive neoplasias (8, 9) as a tumor-specific prosenescence therapy (10, 11). Furthermore, the concept that continuous E6/E7 Author contributions: K.H.-S. and F.H.-S. designed research; K.H.-S., F.B., C.L., J.B., L.J., and A.M. performed research; F.R., P.V., and M.D. analyzed data; and F.H.-S. wrote the paper. expression is essential for the growth of HPV-positive tumor cells The authors declare no conflict of interest. implies that the two viral proteins represent attractive targets for This article is a PNAS Direct Submission. K.M. is a Guest Editor invited by the Editorial immunotherapy, because E6/E7 synthesis cannot be down-regulated Board. as an evasion mechanism (12, 13). 1To whom correspondence should be addressed. Email: [email protected]. – Many cancers are characterized by low O2 concentrations (14 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 16). Hypoxia, usually defined as tissue O2 concentration <1.5% 1073/pnas.1615758114/-/DCSupplemental.

E990–E998 | PNAS | Published online January 23, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1615758114 Downloaded by guest on October 3, 2021 AB PNAS PLUS

CD MICROBIOLOGY

Fig. 1. Repression of HPV E6/E7 oncogene expression under hypoxia. (A) HPV18-positive HeLa and SW756 cells and HPV16-positive SiHa and CaSki cells were

cultured for 24 h at the indicated O2 concentrations. Shown are immunoblots of HIF-1α (hypoxia-linked marker), HPV16/18 E6, HPV16/18 E7, total Rb, phosphorylated Rb (P-Rb; Ser807/811), p53, and p21 protein expression. β-actin served as a loading control. (B) Normoxic cells were transfected with E6/E7-targeting siRNAs (si16E6/E7 or si18E6/E7) or control siRNA (siContr-1), and protein expression was analyzed by immunoblotting. (C) E6/E7 mRNA expression under normoxia

(21% O2) or hypoxia (1% O2). Indicated are the mean E6/E7 transcript levels of at least five independent experiments measured by qRT-PCR after 24 h under hypoxia, for each cell line relative to the corresponding E6/E7 transcript levels under normoxia (set at 1.0). SDs are indicated. Asterisks above columns indicate statistically significant differences from normoxic cells (***P < 0.001). (D) Time course of hypoxia-linked E6/E7 repression. (Upper) Measurements of transcript levels by qRT-PCR. SDs of technical replicates are indicated (n = 3). (Lower) Accompanying analyses of protein levels by immunoblot. β-actin served as a loading control. (E) HPV-positive cancer cells were grown in medium containing 0 mM, 5.5 mM, or 25 mM glucose. The presence (+)orabsence(−) of FCS in the medium is

indicated. Cells were cultured under normoxia (21% O2)orhypoxia(1%O2). Shown are immunoblots of HPV16/18 E6, HPV16/18 E7, and HIF-1α levels. β-actin served as a loading control.

Hoppe-Seyler et al. PNAS | Published online January 23, 2017 | E991 Downloaded by guest on October 3, 2021 HPV oncogenes and the host cell machinery, which also has im- HPV-positive cancer cells, with cell numbers reaching a plateau plications for the clinical behavior of HPV-positive cancers. after 24 h (Fig. 2A). Of note, viral E6/E7 expression was reinduced in HPV-positive cancer cells on reoxygenation, at both the mRNA Results and protein levels (Fig. 2B). During these time course experi- E6/E7 and p53 Expression in Hypoxic HPV-Positive Cancer Cells. HPV18- ments, p53 protein levels showed a biphasic regulation, increasing positive (HeLa, SW756) and HPV16-positive (SiHa, CaSki) cervical soon after reoxygenation but decreasing again at later time points cancer cells were cultured at 21% O2 (“normoxia”), 3% O2,or1% when E6 protein levels began to increase (Fig. 2B). The reac- O2 (“hypoxia”). Interestingly, although a reduction of E6 and E7 tivation of E6/E7 expression was linked to the reinduction of cell protein expression was detectable at 3% O2, we observed a dra- proliferation (Fig. 2C). matic drop at 1% O2 in all HPV-positive cell lines (Fig. 1A). The The reversibility of the hypoxia-linked growth arrest of HPV- efficacy of hypoxic E6/E7 repression was comparable to the ex- positive cancer cells indicates that the cells do not senesce, al- perimental down-regulation of E6/E7 by potent RNA interference though E6/E7 is efficiently repressed. Concordantly, and in contrast (RNAi) under normoxia (Fig. 1B). E6/E7 repression under hyp- to RNAi-mediated E6/E7 repression under normoxia (Fig. 2D, oxia was also detectable at the mRNA level (Fig. 1C), as shown by Right), we observed that hypoxic HPV-positive cancer cells lacked qRT-PCR analyses measuring the amounts of all three transcript the typical morphological signs of senescence (e.g., cell enlarge- classes coding for HPV16 or HPV18 E6/E7 (5). Kinetic analyses ment, flattening, long cytoplasmic projections) and did not stain indicated that the down-regulation of E6/E7 mRNA and protein positive for the senescence marker senescence-associated β-galac- expression started at ∼9–12 h under hypoxia (Fig. 1D). tosidase (SA-β-Gal) (Fig. 2D, Left). HPV-positive cells treated with To gain insight into the underlying molecular mechanism, we E6/E7 inhibitory siRNAs in combination with hypoxia also escaped analyzed candidate pathways that can mediate the cellular re- from senescence (Fig. S3). sponse to hypoxia. The hypoxia-induced factors HIF-1α and HIF- 2α are major coordinators of this process, by modulating the Senescence Induction on E6/E7 Inhibition Requires mTOR Signaling, transcription of a broad array of target genes (22). We did not find Which Is Impaired Under Hypoxia. To decipher the mechanism un- experimental evidence linking HIF-1α or HIF-2α induction to E6/ derlying this differential regulation in hypoxic and normoxic HPV- E7 repression, however, given that HIF mimetics do not repress positive cancer cells, we first addressed the question of which cel- E6/E7 under normoxia and that silencing of HIF-1α or HIF-2α, lular pathway is critical for senescence induction on E6/E7 alone or in combination, does not affect hypoxia-induced E6/E7 repression under normoxia. The mTOR pathway emerged as a down-regulation (Fig. S1). possible candidate, given that mTOR signaling can support senes- Hypoxia also can modulate gene expression by affecting carbo- cence induction and can be inhibited by hypoxia in some, but not hydrate metabolism via HIF-dependent (22) or HIF-independent all, cell types (27). (23, 24) routes. Therefore, we cultured HPV-positive cells in me- We found that hypoxia inhibits mTOR signaling in HPV-positive dium devoid of glucose (0 mM), medium containing physiological cancer cells, as indicated by the quantitative reduction of the serum glucose levels (5.5 mM; 100 mg/dL), and medium containing phosphorylated forms of substrates of the mTOR pathway, in- unphysiologically high glucose concentrations (25 mM; 450 mg/dL). cluding p70-S6 kinase (P-S6K), S6 ribosomal protein (P-S6), and We also tested whether E6/E7 repression depends on serum in the eukaryotic initiation factor 4E-binding protein 1 (P-4E-BP1) medium. Neither glucose nor serum was required for hypoxic (Fig. 3A, Left). The effect is glucose-sensitive and can be coun- E6/E7 repression (Fig. 1E). Of note, however, the inhibitory effect teracted by a high glucose (25 mM) supply (Fig. S4A). The hypoxic of hypoxia on E6/E7 expression was strongly impaired at 25 mM impairment of mTOR signaling is not a peculiarity of HPV-positive glucose (Fig. 1E), indicating that a high glucose supply can effi- cancer cells, and was also detected in a panel of HPV-negative ciently counteract hypoxia-mediated E6/E7 repression. cancer cells under the same experimental conditions (Fig. S4B). In The HPV E6 and E7 oncoproteins target the p53 and pRb tumor contrast to the situation in hypoxic cells, when E6/E7 is inhibited suppressor proteins, respectively, for inactivation (2). RNAi-medi- under normoxia by RNAi, P-S6K and P-S6 concentrations were not ated E6/E7 repression under normoxia resulted in decreased diminished, with P-S6 amounts even increasing in HeLa and SiHa amounts of phosphorylated pRb (Fig. 1B). This regulation was (Fig. 3A, Right). Only P-4E-BP1 levels were somewhat reduced similar in three of four tested cervical cancer cell lines when E6/E7 (Fig. 3A, Right). Collectively, these findings raise the possibility that amounts were down-regulated under hypoxia; the sole exception mTOR activity is critical for senescence induction on E6/E7 in- was HeLa cells, in which phosphorylated pRb levels remained hibition in normoxic HPV-positive cancer cells. largely unaffected (Fig. 1A). Of note, however, we found profound To directly address this question, we silenced E6/E7 ex- differences in the regulation of p53. RNAi-mediated E6/E7 repression in normoxic HPV-positive cancer cells by transient pression under normoxia resulted in strong quantitative increases in transfection with siRNAs in either the absence or the presence p53 protein, as expected from its interference with E6-mediated p53 of the chemical mTOR inhibitors KU-0063794 and rapamycin. degradation (25), as well as in p21 induction, representing a tran- To exclude the possibility that untransfected cells (in which scriptional target gene for p53 (Fig. 1B). In contrast, despite effi- E6/E7 is not silenced by RNAi) can form colonies, we also cient E6/E7 repression, p53 protein levels did not increase in any of treated cells with nocodazol to eliminate cells that were not the investigated HPV-positive cancer cell lines under hypoxia, but arrested (28, 29). SA-β-Gal analyses indicated that HPV-posi- remained unaffected or even decreased (Fig. 1A). We observed only tive cancer cells, in which E6/E7 expression was silenced alone, marginal and nonsignificant effects on p53 mRNA levels (Fig. S2A). became senescent (Fig. 3B, Upper Left). In contrast, the con- Treatment with Nutlin-3, a potent inhibitor of the MDM2/p53 in- comitant treatment with either mTOR inhibitor resulted in the teraction (26), did not markedly increase p53 protein levels in HPV- outgrowth of cells that do not stain for SA-β-Gal (Fig. 3B, positive cancer cells, in contrast to the response of HPV-negative Center Left and Lower). These findings show that mTOR in- HCT116 colon cancer cells, which showed a strong up-regulation of hibitors enable the escape of HPV-positive cancer cells from p53 concentrations under the same experimental conditions (Fig. senescence, despite efficient E6/E7 repression (Fig. 3C). S2B). Consistent with the lack of p53 induction in hypoxic HPV- This idea is further corroborated by results of colony-formation positive cancer cells, p21 expression was not increased (Fig. 1A). assays (CFAs) following transient transfection with E6/E7- inhibitory siRNAs. We found that the concomitant exposure to Hypoxic E6/E7 Repression Does Not Result in Senescence. We next rapamycin or KU-0063794 led to a strong increase in the colony- examined the phenotypic consequences of hypoxia-induced E6/E7 formation capacity of HPV-positive cancer cells (Fig. 3B, Right). repression. We found that hypoxia inhibits the proliferation of The mTOR inhibitors did not reinduce HPV E6 or E7 expression

E992 | www.pnas.org/cgi/doi/10.1073/pnas.1615758114 Hoppe-Seyler et al. Downloaded by guest on October 3, 2021 (Fig. 3C) and induced the expected effects on the phosphorylation that mTOR signaling is critical for the induction of senescence on PNAS PLUS of mTOR substrates (30, 31), with P-4E-BP1 being largely re- E6/E7 repression in normoxic HPV-positive cancer cells. sistant to rapamycin treatment and KU-0063794 leading to re- We next investigated whether the lack of senescence induction ductions in P-4E-BP1 and P-S6K (Fig. 3C). These results show observed in hypoxic HPV-positive cancer cells is due to impaired mTOR signaling. Many different pathways have been implicated in the weakening of mTOR signaling under hypoxia (32), including stimulation of the HIF-1 target gene REDD1 (regulated in devel- ACopment and DNA damage responses 1), which interferes with mTOR signaling by activating the mTOR inhibitor TSC2 (tuberous sclerosis complex 2) (33, 34). We observed that hypoxia leads to the up-regulation of REDD1 expression in HeLa cells (Fig. S5A), raising the possibility that the mTOR-inhibitory REDD1/TSC2 axis plays a critical role in the blocking of senescence in hypoxic HPV- positive cancer cells. We generated shRNAs that blocked TSC2 or REDD1 expression (Fig. S5B) and found that they stimulated mTOR signaling under hypoxia, as shown by the increased amounts of P-S6K and P-S6 at 1% O2 (Fig. 3D and Fig. S5C), whereas E7 oncoprotein expression was not reinduced (Fig. 3D). Under our experimental conditions, p53 levels did not increase in HPV-positive cancer cells on stimulation of mTOR signaling at 1% O2 (Fig. S6A) and did not decrease on inhibition of mTOR signaling at 21% O2 (Fig. S6B). B Of note, stimulation of the mTOR pathway in hypoxic HPV- positive cancer cells led to the emergence of cells staining positive for SA-β-Gal (Fig. 3E, Left and Fig. S5D), indicating the induction of senescence. Accordingly, the cells showed decreased colony- formation capacity when switched to normoxic culture conditions

(Fig. 3E, Right). Collectively, these results indicate that hypoxic MICROBIOLOGY HPV-positive cancer cells, in which E6/E7 expression is down- regulated, evade senescence owing to the concomitant impairment of mTOR signaling that occurs, at least in part, via stimulation of the inhibitory REDD1/TSC2 axis.

Hypoxia Blocks Chemotherapy-Induced Senescence in HPV-Positive Tumor Cells. There is increasing evidence that, along with apoptosis, senescence induction in tumor cells is a major mechanism through which chemotherapeutic agents can exert their anticancer effects (10, 35). Therefore, we also tested the impact of chemo- therapeutics on the senescence regulation of HPV-positive cancer cells, dependent on their O2 supply. As shown in Fig. 4A, Left, treatment of normoxic HPV16-positive D SiHa cells with etoposide efficiently induced senescence. Concom- itant treatment with rapamycin or KU-0063794 allowed evasion of the cells from etoposide-induced senescence, as indicated by the outgrowth of SA-β-Gal–negative cells. This finding is corroborated by CFAs showing that the simultaneous application of mTOR inhibitors enhanced the colony-forming capacity of HPV-positive cells (Fig. 4A, Right) compared with etoposide treatment alone. We next analyzed whether hypoxia can exert a similar detri- mental effect on CT-induced senescence in HPV-positive cancer cells as was noted for chemical mTOR inhibitors. We observed that hypoxia allowed the cells to escape from etoposide-induced senescence (Fig. 4B, Left) and resulted in increased colony-formation Fig. 2. Hypoxia-induced growth inhibition and E6/E7 repression are reversible capacity (Fig. 4B, Right). This response is consistent for all HPV- on reoxygenation. (A) HeLa and SiHa cells were cultured for the indicated time positive cancer cell lines examined (Fig. 4C) and not specific for periods at 21% O2 or at 1% O2, and relative cell numbers were determined. For each cell line, initial cell numbers (time point 0) were set at 1.0. SDs of etoposide, but was also detected for another tested chemothera- biological replicates are indicated (n = 4). (B, Upper) qRT-PCR analyses of E6/E7 peutic drug, doxorubicin (Fig. 4D).

mRNA levels under normoxia (N; 21% O2) and hypoxia (H; 1% O2)(bright We then investigated whether the evasion of hypoxic HPV-positive columns), and on reoxygenation of hypoxic cells for the indicated time periods tumor cells from etoposide-induced senescence is due to impaired (dark columns). SDs of technical replicates are indicated (n = 3). (B, Lower) mTOR signaling. We found that REDD1- or TSC2-inhibitory Accompanying immunoblot analyses of E6, E7, p53, and HIF-1α levels. β-actin shRNAs reduced the outgrowth of SA-β-Gal–negative cells under served as a loading control. (C) Cells were incubated for 72 h at 1% O2.Sub- hypoxia (Fig. 4E, Left and Fig. S5E), indicating that fewer hypoxic sequently (time point 0), the cells were cultured at 21% O2 for the indicated HPV-positive cells can evade etoposide-induced senescence when time periods, and cell numbers were determined. SDs of biological replicates mTOR signaling is stimulated. Consistently, etoposide treatment are indicated (n = 3). (D, Left) Cells were cultured under normoxia (21% O )or 2 of hypoxic cells, in which mTOR signaling is increased, results in a hypoxia (1% O2) for 72 h and the stained for expression of the senescence marker SA-β-Gal. (Scale bar: 200 μm.) (D, Right) Cells were cultured under reduced colony-formation capacity when the cells are switched to normoxia, endogenous E6/E7 expression was silenced by RNAi, and cells were normoxic culture conditions (Fig. 4E, Right). Taken together, these stained for SA-β-Gal expression at 72 h after transfection. results indicate that intact mTOR signaling is crucial not only for

Hoppe-Seyler et al. PNAS | Published online January 23, 2017 | E993 Downloaded by guest on October 3, 2021 HeLa SiHa CaSki

A HeLa SW756 SiHa CaSki siContr-1 si18E6/E7 siContr-1 si16E6/E7 21 3 1 21 3 1 21 3 1 21 3 1 % O2 siContr-1 si16E6/E7 P-S6K P-S6K

S6K S6K

P-S6 P-S6

P-4E-BP1 P-4E-BP1

4E-BP1 4E-BP1

Vinculin Vinculin

B HeLa SiHa HeLa SiHa si18E6/E7 si16E6/E7 si18E6/E7 si16E6/E7 C HeLa SiHa

- si18E6/E7 si16E6/E7 siContr-1 - R KU siContr-1 - R KU E7

P-S6K

Rapamycin S6K

P-4E-BP1

4E-BP1

KU-0063794 ß-Actin

Tx split wash SA-ß-Gal CFA

031day 2 9 15 20 HeLa HeLa R or KU E nocodazole 21% O2 1% O2 21% O2 1% O2

D HeLa pSUPER shcont-1 shContr-1 shREDD1-1 shTSC2-1 shContr-1 shREDD1-2 -1 shREDD1-1 21 1 21 1 21 1 21 1 % O2 P-S6K

S6K shREDD1 -2

P-S6 shTSC2-1

18E7 Tx split SA-ß-Gal CFA

Vinculin day031 2 4 7 11 shTSC2-1 1% O2

Fig. 3. Senescence induction on E6/E7 repression in HPV-positive cancer cells depends on mTOR signaling, which is impaired under hypoxia. (A, Left) Cells were

cultured under different O2 concentrations, as indicated. Immunoblot analyses of phosphorylated S6K (P-S6K), total S6K (S6K), phosphorylated S6 (P-S6), phosphorylated 4E-BP1 (P-4E-BP1) and total 4E-BP1 (4E-BP1). Vinculin served as a loading control. (A, Right) Immunoblot analyses of mTOR substrates on RNAi-mediated E6/E7 repression in normoxic HPV-positive cancer cells. (B, Left) Senescence assays (SA-β-Gal staining), in the absence (−) or the presence of the mTOR inhibitors rapamycin or KU-0063794. (Scale bar: 200 μm.) (B, Right) Colony-formation assays of HPV-positive cancer cells, in the absence (−) or presence of rapamycin or KU- 0063794. Colonies were visualized with crystal violet. Scheme below: Treatment protocol, further detailed in the text. Tx, transfection. (C) Normoxic HPV-positive cancer cells were transfected with E6/E7-inhibitory siRNAs in either the absence (−) or presence of rapamycin (R) or KU-0063794 (KU). The levels of HPV16/18 E6 and E7, P-S6K, S6K, P-4E-BP1, and 4E-BP1 were determined by immunoblotting. siContr-1, control siRNA. β-actin served as a loading control. (D) HeLa cells expressing shREDD1-1,

shREDD1-2, or shTSC2-1 were cultured at 21% or 1% O2 for 24 h, after which levels of P-S6K, S6K, P-S6 and HPV18 E7 were determined by immunoblotting. Vinculin served as a loading control. (E) HeLa cells were transfected with shRNA expression vectors for shREDD1-1, shREDD1-2, or shTSC2-1 and cultured under normoxia or hypoxia. (Left) Senescence assays (SA-β-Gal staining) of cells subsequently cultured under normoxia. (Scale bar: 200 μm.) (Right) Corresponding CFAs. Control cells were transfected with empty vector (pSUPER) or with a vector expressing shContr-1. Scheme below: Treatment protocol, further detailed in the text. Tx, transfection.

senescence induction in HPV-positive cancer cells on endogenous by immunohistochemistry. We observed prominent tumor areas E6/E7 repression, but also for their response toward external with strong CA IX staining in 9 of the 17 cancers. Notably, and prosenescent stimuli, such as chemotherapeutic drugs. consistent with the in vitro data presented above, we found that these tumor areas invariably exhibited an inverse correlation be- Inverse Correlation Between Expression of HPV E7 and Carbonic tween HPV16 E7 and CA IX expression levels (Fig. 5A). Anhydrase IX in Cervical Cancer. Finally, we investigated the in To allow a more detailed investigation of the relative spatial vivo expression of the HPV E7 oncoprotein in relation to the distribution of the antigens of interest, we prepared multicolor hypoxia-linked marker carbonic anhydrase IX (CA IX) (36) in immunofluorescence stains for CA IX, E7, CD34 (staining micro- HPV16-positive squamous cell cervical cancer specimens (n = 17) vascular endothelium), and Ki-67 (proliferation marker), using

E994 | www.pnas.org/cgi/doi/10.1073/pnas.1615758114 Hoppe-Seyler et al. Downloaded by guest on October 3, 2021 PNAS PLUS

A split SA-ß-Gal CFA C HeLa SW756 CaSki MRI-H-186 2 day031 2 10 13 15 Etoposide or Doxorubicin 21% O R, KU or 1% O2 2 SiHa Etoposide Etoposide 1% O

- D HeLa SW756 CaSki MRI-H-186 SiHa 2 21% O 2 Doxorubicin Rapamycin 1% O

HeLa Etoposide KU-0063794 21% O 1% O E HeLa 2 2 Etoposide

21% O2 1% O2 B SiHa pSUPER 2 shContr-1 shContr-1 21% O -1 MICROBIOLOGY Etoposide 2 shREDD1 1% O -2 shTSC2-1 shREDD1-1 shTSC2-1

Fig. 4. CT-induced senescence in HPV-positive cancer cells depends on mTOR signaling and is counteracted by hypoxia. (A) SiHa cells were treated under normoxia with etoposide, in either the absence (−) or the presence of rapamycin or KU-0063794. (Left) Senescence assays (SA-β-Gal staining). (Scale bar: 200 μm.) (Right) CFAs. Scheme above: Treatment protocol for Fig. 4 A–D, further detailed in the text. (B) SiHa cells were treated for 48 h with etoposide under normoxia or hypoxia, and subsequently cultured under normoxia. (Left) Senescence assays (SA-β-Gal staining). (Scale bar: 200 μm.) (Right) CFAs. (C and D)

Normoxic and hypoxic HPV16- and HPV18-positive cancer cell lines were exposed to etoposide (C) or doxorubicin (D) at 21% or 1% O2, and subsequently analyzed by CFAs under normoxia. (E) HeLa cells were transfected with shRNA expression vectors for shREDD1-1, shREDD1-2, or shTSC2-1 and cultured under normoxia or hypoxia. (Left) Senescence assays (SA-β-Gal staining) of cells subsequently cultured under normoxia. (Scale bar: 200 μm.) (Right) Corresponding CFAs. Control cells were transfected with empty vector (pSUPER) or with a vector expressing shContr-1. Treatment protocol corresponds to the scheme indicated in Fig. 3E, but with additional etoposide treatment at days 2–4.

DAPI as a counterstain for cell nuclei. Again, we regularly detected response to E6/E7 repression in normoxic HPV-positive cancer an inverse correlation between CAIXandE7proteinexpression cells requires intact mTOR signaling, which is impaired in hypoxic (Fig. 5B). Costaining for CD34 revealed that E7 is expressed pre- HPV-positive cancer cells via the mTOR inhibitory REDD1/TSC2 dominantly in the vicinity of blood vessels, whereas CA IX is axis. The ability of HPV-positive cancer cells to induce senescence predominantly expressed more distant from blood vessels (Fig. 5B). under hypoxia can be restored by experimental reactivation of Moreover, in all investigated cervical cancer specimens, we de- mTOR signaling. Collectively, these findings indicate that hypoxic tected CA IX-positive/E7-negative regions in which expression of conditions allow HPV-positive cancer cells to withdraw from the Ki-67 is strongly reduced or absent (dotted lines in Fig. 5B; another selection pressure to continuously express E6/E7 without un- example of a cervical cancer specimen from a different patient is dergoing an irreversible growth arrest, suggesting that our current provided in Fig. S7), indicating the presence of hypoxic tumor areas conception of the cross-talk between the HPV oncogenes and the with little or no cell proliferation. host cell is likely too simplistic. The evasion from senescence by hypoxic HPV-positive cancer cells could be crucial for the patho- Discussion genesis of HPV-positive cancers, in that it allows escape from a In this study, we found that hypoxic HPV-positive cancer cells can major tumor-suppressive defense mechanism of the cell (7). convert to a state of dormancy in which they efficiently shut off Our findings in this study have clinical implications as well E6/E7 expression, induce a proliferative halt, but do not senesce. (Fig. 6). The reversibility of hypoxia-induced growth inhibition The cells are reactivated when they regain access to improved O2 may contribute to cancer recurrence when dormant HPV-positive supply, viral oncogene expression is reinduced, and proliferation is cancer cells regain access to an increased O2 supply, which can resumed. This phenotype differs fundamentally from the behavior occur, for example, following tumor shrinkage after therapy (37) of HPV-positive cancer cells under normoxic conditions, where E6/ or on neovascularization (38). Moreover, the hypoxia-induced E7 repression results in rapid induction of senescence. Mechanis- proliferative halt of HPV-positive cancer cells likely provides them tically, we show that these discrepancies can be attributed to dif- with increased resistance to CT, which preferentially attacks pro- ferences in mTOR signaling. We found that the senescence liferating cells. In line with these considerations, hypoxia is linked

Hoppe-Seyler et al. PNAS | Published online January 23, 2017 | E995 Downloaded by guest on October 3, 2021 A stimulus. We found that mTOR inhibitors can also interfere with CA IX HPV16 E7 the prosenescent activity of CT. Currently, mTOR inhibitors are under investigation in clinical studies as anticancer agents and are showing growth-inhibiting effects in preclinical models, including xenografts of HPV-positive cancer cells (41–43). The antitumorigenic effects of mTOR inhibitors in the clinic have often been unsatisfying when given as a monotherapy, however (44, 45). Increasingly, mTOR inhibitors are being used in combination with CT, to sensitize tumor cells toward chemotherapeutic agents (46). Given that the prosenescent activity of CT is considered important for its anticancer effects (10, 35), this use is difficult to reconcile with our data and data of others (47) showing that mTOR in- B hibitors can block CT-induced senescence. A possible explanation for this discrepancy is that senescence induction by CT could also have undesired effects, in that senescent cells within the tumor microenvironment (including stromal fibroblasts) secrete protumorigenic factors [Senescence-Associated Secretory Phenotype (SASP)] (7). Indeed, recent studies have indicated that the CT- sensitizing effect of mTOR inhibitors is linked to their ability to interfere with secretion of major components of the SASP of stromal cells (48, 49). Thus, the biological effects of mTOR inhibitors are likely to be complex, and their efficacy in cancer therapy may be determined by the balance between potentially protumorigenic (blocking the senescence response of cancer cells) and antitumorigenic (cytostasis, interference with the SASP, inhibition of angiogenesis) (50) responses. Our findings also provide a foundation for future studies in several related areas. First, what is the mechanism that prevents the reconstitution of p53 under hypoxia-induced E6/E7 repression? Under normoxia, the p53 levels in HPV-positive cancer cells are Fig. 5. Negative correlation between E7 and CA IX expression in tissue dependent on E6-mediated proteolytic p53 degradation (51). Our specimens from patients with cervical cancer. (A) Representative immuno- results indicate that this control mechanism is uncoupled under histochemical analysis of an HPV16-positive cervical cancer, stained for the hypoxia, in that E6 repression no longer results in increased p53 expression of the hypoxia-linked marker CA IX and HPV16 E7. (Scale bar: levels. Hypoxic HPV-positive cells do not appear to switch from 200 μm.) (B) Multiplex immunofluorescence staining of the tumor depicted in Fig. 5A. Nuclei were counterstained with DAPI (blue). (Upper, Left)Ex- pression of CD34 (red) and E7 (orange). (Upper, Right) Expression of CD34 (red) and CA IX (green). (Lower, Left) Expression of CD34 (red), E7 (orange) and CA IX (green). (Lower, Right) Expression of CD34 (red), CA IX (green) and Resistance to Ki67 (white). (Scale bar: 100 μm.) Note the autofluorescence of red blood Hypoxia cells in the red channel. See also Fig. S7. E6/E7 inhibitors

to a worsened clinical prognosis, as well as to an increased ther- E6/E7 Immune apeutic resistance of HPV-positive cancers (15, 16, 20). evasion Furthermore, it is conceivable that the hypoxic E6/E7 repression mTOR reduces the synthesis and, subsequently, the presentation of viral antigens on the cell surface. In concert with the general immunosuppressive effects of hypoxia (39), this may help HPV-positive cancer cells evade the patient’s immune system in hypoxic tumor Resistance to Reversible Cancer regions. The latter scenario could also represent a major obstacle chemotherapy growth arrest recurrence for immunotherapeutic approaches targeting E6/E7-derived anti- (no senescence) gens, providing a possible molecular explanation as to why the success of immunotherapy for the treatment of HPV-positive Reoxygenation cancers has been rather limited to date (13, 40). Our findings also raise concerns about the strategy to block E6/E7 expression as a prosenescent therapeutic approach, in that Fig. 6. Implications of hypoxia-linked alterations in HPV-positive cancer cells. Hypoxia results in E6/E7 repression and inhibition of cellular pro- E6/E7 inhibitors could be ineffective in hypoxic HPV-positive liferation of HPV-positive cancer cells. The concomitant interference with cancer cells where E6/E7 expression is shut down and mTOR mTOR signaling by hypoxia allows the cells to evade senescence. The growth signaling is impaired. However, these considerations do not pre- inhibition under hypoxia contributes to the resistance of HPV-positive cancer clude a therapeutic use of E6/E7 inhibitors, because they may be cells toward CT. The ability of hypoxic HPV-positive cancer cells to block effective in better-oxygenated regions of HPV-positive tumors and E6/E7 expression without undergoing senescence also provides therapeutic could reduce tumor size. Moreover, our data show that the resistance toward strategies aiming at E6/E7 inhibition. The repression of regrowth of dormant HPV-positive cancer cells on reoxygenation E6/E7 antigen synthesis, together with the immunosuppressive effects of hypoxia, support evasion of hypoxic HPV-positive cancer cells from the host’s is linked to the reinduction of viral E6/E7 oncogene expression, immune response and protects against immunotherapeutic approaches which may then allow targeting of these cells by E6/E7 inhibitors. targeting E6/E7. Owing to the reversibility of hypoxia-linked growth in- The critical significance of the mTOR pathway in HPV-positive hibition, dormant hypoxic HPV-positive cells could serve as a reservoir for cells is not limited to E6/E7 repression acting as a prosenescent tumor recurrence on reoxygenation.

E996 | www.pnas.org/cgi/doi/10.1073/pnas.1615758114 Hoppe-Seyler et al. Downloaded by guest on October 3, 2021 E6-dependent to MDM2-dependent p53 degradation, given that Schweizer, Arbor Vita Corporation); anti-p53 (sc-126) and anti-vinculin (sc- PNAS PLUS treatment with Nutlin-3 did not appreciably reincrease p53 levels. 73614) (Santa Cruz Biotechnology); anti-p21 (556431) and anti–HIF-1α (610959) In addition, p53 activities may be further blunted by the impair- (BD Biosciences); anti–HIF-2α (NB100-122; Novus Biologicals); and anti-Rb – – ment of p53 transactivation function under hypoxia (52, 53). (9309), anti phospho-Rb (Ser807/811) (9308), anti phospho-S6 (Ser235/236) (2211), anti–4E-BP1 (9452), anti–phospho-4E-BP1 (Ser65) (9451), anti-p70S6K Second, it will be interesting to further decipher in detail the (9202), and anti–phospho-p70S6K (Thr389) (9234) (Cell Signaling Technology). molecular mechanism of hypoxia-linked E6/E7 repression. We The following HRP-conjugated secondary antibodies were used: anti-mouse have shown that high glucose concentrations (25 mM), which can IgG (W402), anti-chicken IgY (G1351), and anti-goat IgG (V8051) (Promega), be achieved in the blood of individuals with severe uncontrolled and anti-rat IgG (112035003; Dianova). diabetes, efficiently counteract hypoxic E6/E7 repression. The basis of glucose-linked effects on gene expression is complex and in- RNA Extraction and qRT-PCR. All qRT-PCR analyses were performed at least three completely understood, but can involve epigenetic mechanisms as times, in duplicate. For time courses, representative experiments (qRT-PCR well as specific transcription factors, including MondoA/ChREBP- performed in triplicate) are depicted along with corresponding protein analyses. Mlx, NF-κB, c-Myc, and SP1 (54, 55). Third, it will be important to RNA extraction and qRT-PCR conditions are detailed elsewhere (5). Forward study how E6/E7 repression under hypoxia influences viral antigen (fwd) and reverse (rev) primer sequences (Eurofins MWG) were as follows: 18E6/ E7 fwd: 5′-ATGCATGGACCTAAGGCAAC-3′; 18E6/E7 rev: 5′-AGGTCGTCTGCT- presentation on HPV-positive cancer cells and thereby may sup- GAGCTTTC-3′; 16E6/E7 fwd: 5′-CAATGTTTCAGGACCCACAGG-3′; 16E6/E7 rev: port their escape from immune defense mechanisms of the host. 5′-CTCACGTCGCAGTAACTGTTG-3′; REDD1 fwd: 5′-CCTCACCATGCCTAGCCTTT-3′; In conclusion, the results of the present study show that the cross- REDD1 rev: 5′-GTAAGCCGTGTCTTCCTCCG-3′; TSC2 fwd: 5′-TCCTCGACCA- talk between the HPV oncogenes and the host cell machinery, as GATCCCATCA-3′;TSC2rev:5′-GCCATGCTCATTGGACAGGA-3′; 18S RNA fwd: well as the resulting phenotypic consequences in HPV-positive 5′-CATGGCCGTTCTTAGTTGGT-3′; 18S RNA rev: 5′-ATGCCAGAGTCTCGTTCGTT-3′. −ΔΔ cancer cells, are profoundly dependent on cellular oxygenation Relative RNA quantification was performed using the comparative Ct (2 Ct) levels. The ability of hypoxic HPV-positive cancer cells to induce a method (58). Data are presented as the fold change in gene expression, nor- dormant state, reversibly blocking viral oncogene expression and malized to a reference gene (18S RNA), and relative to a calibrator sample (5). Statistical significance was determined by the two-tailed Student’s t test. P cellular proliferation without undergoing senescence, is likely to < < affect the clinical behavior of HPV-positive cancers and should be values of **P 0.01 and ***P 0.001 were considered significant. considered in the ongoing development of novel treatment strate- CFAs, Senescence Assays, and Cell Counts. For CFAs on RNAi-mediated E6/E7 gies, such as immunotherapy or targeted E6/E7 inhibition. repression (Fig. 3B), cells were transfected with E6/E7-inhibitory siRNAs, in the absence or presence of mTOR inhibitors. At 2 d after transfection, cells were

Materials and Methods split and further grown in the absence or presence of mTOR inhibitors. MICROBIOLOGY Cell Culture, Transfections, Treatments, and Reagents. HPV18-positive HeLa and Starting 24 h after splitting, cells were also treated with nocodazole for 6 d, to SW756 cervical carcinoma cells, and HPV16-positive SiHa, CaSki, and MRI-H-186 eliminate nontransfected proliferating cells while sparing growth-arrested cervical carcinoma cells, as well as HPV-negative C33A cervical cancer cells, cells, following the protocol established by Leontieva et al. (27, 28). Then cells spontaneously immortalized HaCaT keratinocytes, HepG2 hepatoma cells, U2OS were cultured in drug-free medium, fixed, and stained with formaldehyde- osteosarcoma cells, HCT116 and RKO colon cancer cells, and MCF7 breast cancer crystal violet at 20 d after transfection. For analyzing the effects of CT by CFAs cells, were obtained from the tumor bank of the German Cancer Research Center (Fig. 4 A–D), cells were cultured under hypoxia for 24 h and subsequently or from the American Tissue Culture Collection. Cells were certified negative for treated with 5–10 μM etoposide, depending on the cell line, or with 0.2 μM mycoplasma contamination by PCR, and their identities were verified by mul- doxorubicin. The cells were grown for another 48 h under hypoxia, split, and tiplex human cell line authentication. Authenticated cells were frozen in ali- incubated for 10–12 d under normoxia in drug-free medium. Colonies were quots, and after thawing, cells were used in experiments for a maximum of 4 wk. fixed and stained with formaldehyde-crystal violet. Control cells were treated Cells were cultured under normoxia (21% O2,5%CO2), reduced oxygen (3% accordingly, but consistently kept under normoxia. For the analyses of mTOR O2,5%CO2), or hypoxia (1% O2,5%CO2)inDMEM(HeLa,SW756,SiHa,CaSki, inhibitors on the cellular response toward etoposide (Fig. 4A), cells were cul- C33A, HepG2, U2OS, and MCF7), RPMI (MRI-H186 and RKO) or McCoy’s5A tured in the absence or presence of 50 nM rapamycin or 1 μM KU-0063794 for medium (HCT116), supplemented with 10% FCS (Life Technologies), 2 mM 24 h, then also treated with etoposide for 48 h, split, and further processed as L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich). described above. For senescence assays, cells were treated as described for the The standard cell culture medium contained 100 mg/dL glucose (5.5 mM). CFAs, and stained for SA-β-Gal activity, as detailed previously (5), after the The following chemicals were used for treatment: rapamycin (AdipoGen); indicated time periods. All CFAs and senescence assays were performed in-

nocodazole (Merck); etoposide and doxorubicin (Enzo Life Science); CoCl2, dependently at least three times, with consistent results. Viable cell numbers mimosine, DMOG, and KU-0063794 (Sigma-Aldrich); and Nutlin-3 (Cayman were determined by a standard trypan blue technique, using a Countess cell Chemical). siRNAs were chemically synthesized (Life Technologies) or expressed counter (Thermo Fisher Scientific). Cell count experiments were performed at as shRNAs from vector pSUPER as described previously (56). The si/shRNA target least three times, in duplicate. sequences were as follows: HPV18 E6/E7-1: 5′-CCACAACGUCACACAAUGU-3′; HPV18 E6/E7-2: 5′-CAGAGAAACACAAGUAUAA-3′;HPV18E6/E7-3: 5′-UCCAG- Immunohistochemistry and Multiplex Immunofluorescence Staining. Conven- CAGCUGUUUCUGAA-3′; HPV16 E6/E7-1: 5′-CCGGACAGAGCCCAUUACA-3′; tional immunohistochemistry was performed using heat-induced epitope re- HPV16 E6/E7-2: 5′-CACCUACAUUGCAUGAAUA-3′; HPV16 E6/E7-3: 5′-CAACU- trieval, and DakoEnvision technology was used as described previously (59). GAUCUCUACUGUUA-3′;HIF-1α-1: 5′-CUAACUGGACACAGUGUGU-3′;HIF-1α-2: Multicolor immunofluorescence staining was carried out using a modification 5′-CUGAUGACCAGCAACUUGA-3′;HIF-2α-1: 5′-GCGACAGCUGGAGUAUGAA-3′; of the method published by Toth and Mezey (60). In brief, specimens were HIF-2α-2: 5′-CAGCAUCUUUGAUAGCAGU-3′;REDD1-1:5′-GAAGCTGTACAG- dewaxed in two changes of fresh xylene and then rehydrated in a descending CTCGGAA-3′; REDD1-2: 5′-GGAACAGCTGCTCATTGAG-3′; TSC2-1: 5′-GCTCAT- alcohol series. Retrieval of antigenic binding sites was performed by heating CAACAGGCAGTTC-3′ (57); TSC2-2: 5′-CGACGAGTCAAACAAGCCA-3′ (57); specimens in appropriate buffers (Tris/EDTA 10/1 mM, pH 9.0; citrate pH 6.0) in control siRNA, siContr-1: 5′-CAGUCGCGUUUGCGACUGG-3′, containing at least a steamer (FS10; Braun) for 40 min. For the detection of HPV16 E7, a mouse four mismatches to all known human genes. To minimize potential off-target monoclonal antibody, clone hrHPV-E7 5B4K2, was used. All other primary effects, three different siRNAs, each targeting all three HPV18 or HPV16 E6/E7 antibodies were purchased from Abcam (CA IX, ab108352; Ki67, ab16667; transcript classes, were pooled at equimolar concentrations (referred to in the CD34, ab81289) and detected using polymer-based, HRP-conjugated anti- text as si18E6/E7 and si16E6/E7, respectively), as detailed previously (5). Syn- rabbit or anti-mouse SuperPicture reagents (Thermo Fisher Scientific). Tyr- thetic siRNAs were transfected with DharmaFECT I (Thermo Fisher Scientific) at amide conjugates of Alexa Fluor 488 (CA IX), Alexa Fluor 546 (CD 34) (Thermo a final siRNA concentration of 10 nM, and pSUPER plasmids were transfected Fisher Scientific), and Cy5 (PerkinElmer) were used for visualization. Tyramide by calcium phosphate coprecipitation (56). conjugation of ATTO 425 (ATTO-TEC; Siegen) for Ki-67 visualization was carried out according to the method described by Hopman et al. (61). Quenching Immunoblot Analyses. Cellular protein was prepared and analyzed by immu- of residual peroxidase activity between successive rounds of antigen detection noblotting as described previously (5). The following primary antibodies were was achieved by reheating tissue specimens in the appropriate retrieval buf- used: anti–β-actin (A2228; Sigma-Aldrich); anti-HPV18 E7 (E7C); anti-HPV16 E7 fers. Nuclei were counterstained with DAPI, and slides were covered with a (NM2, kind gift from Martin Müller, German Cancer Research Center); anti- coverslip using ProLong Gold mounting medium (Thermo Fisher Scientific) HPV18 E6 (AVC 399) and anti-HPV16 E6 (AVC 843) (kind gift from Johannes and dried overnight. Digital images of the specimens were acquired using a

Hoppe-Seyler et al. PNAS | Published online January 23, 2017 | E997 Downloaded by guest on October 3, 2021 fluorescence-enabled digital Slidescanner equipped with a LED light source Ethics Committee of the Friedrich-Schiller University Jena (reference nos. 0175– and appropriate filter sets at a magnification of 0.325 μm/pixel (Pannoramic 02/00 and 2174–12/07). Confocal; 3D Histech). All patients provided written informed consent to use their biopsy material ACKNOWLEDGMENTS. We thank Julia A. Braun, Dr. Annette Kopp-Schneider, for further molecular analyses to be conducted in the Jena University Hospital and Dr. Martin Scheffner for discussions. This work was supported by the Wilhelm and in collaboration with academic partners. This study was approved by the Sander-Stiftung (Grant 2015.137.1) and the Deutsche Krebshilfe (Grant 112132).

1. zur Hausen H (2002) Papillomaviruses and cancer: From basic studies to clinical ap- 32. Wouters BG, Koritzinsky M (2008) Hypoxia signalling through mTOR and the un- plication. Nat Rev Cancer 2(5):342–350. folded protein response in cancer. Nat Rev Cancer 8(11):851–864. 2. Howley PM, Schiller JT, Lowy DR (2013) Papillomaviruses. Fields Virology,edsKnipeDM, 33. Brugarolas J, et al. (2004) Regulation of mTOR function in response to hypoxia by Howley PM (Lippincott, Williams & Wilkins, Philadelphia), 6th Ed, pp 1662–1703. REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev 18(23):2893–2904. 3. American Cancer Society (2015) Cancer Facts and Figures 2015 (American Cancer So- 34. Reiling JH, Hafen E (2004) The hypoxia-induced paralogs Scylla and Charybdis inhibit ciety, Atlanta, GA). growth by down-regulating S6K activity upstream of TSC in Drosophila. Genes Dev 4. Goodwin EC, et al. (2000) Rapid induction of senescence in human cervical carcinoma 18(23):2879–2892. cells. Proc Natl Acad Sci USA 97(20):10978–10983. 35. Ewald JA, Desotelle JA, Wilding G, Jarrard DF (2010) Therapy-induced senescence in 5. Honegger A, et al. (2015) Dependence of intracellular and exosomal microRNAs on viral cancer. J Natl Cancer Inst 102(20):1536–1546. E6/E7 oncogene expression in HPV-positive tumor cells. PLoS Pathog 11(3):e1004712. 36. Olive PL, et al. (2001) Carbonic anhydrase 9 as an endogenous marker for hypoxic cells 6. Wells SI, et al. (2000) Papillomavirus E2 induces senescence in HPV-positive cells via in cervical cancer. Cancer Res 61(24):8924–8929. pRB- and p21(CIP)-dependent pathways. EMBO J 19(21):5762–5771. 37. Seiwert TY, Salama JK, Vokes EE (2007) The concurrent chemoradiation paradigm— 7. Campisi J (2013) Aging, cellular senescence, and cancer. Annu Rev Physiol 75:685–705. general principles. Nat Clin Pract Oncol 4(2):86–100. 8. Hellner K, Münger K (2011) Human papillomaviruses as therapeutic targets in human 38. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature cancer. J Clin Oncol 29(13):1785–1794. 407(6801):249–257. 9. Hoppe-Seyler F, Hoppe-Seyler K (2011) Emerging topics in human tumor virology. Int J 39. Noman MZ, et al. (2015) Hypoxia: A key player in antitumor immune response. Am J Cancer 129(6):1289–1299. Physiol Cell Physiol 309(9):C569–C579. 10. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP (2011) Pro-senescence therapy for 40. Skeate JG, Woodham AW, Einstein MH, Da Silva DM, Kast WM (2016) Current ther- cancer treatment. Nat Rev Cancer 11(7):503–511. apeutic vaccination and immunotherapy strategies for HPV-related diseases. Hum 11. Acosta JC, Gil J (2012) Senescence: A new weapon for cancer therapy. Trends Cell Biol Vaccin Immunother 12(6):1418–1429. 22(4):211–219. 41. Molinolo AA, et al. (2012) mTOR as a molecular target in HPV-associated oral and 12. Nizard M, et al. (2013) Immunotherapy of HPV-associated head and neck cancer: cervical squamous carcinomas. Clin Cancer Res 18(9):2558–2568. Critical parameters. OncoImmunology 2(6):e24534. 42. Coppock JD, et al. (2013) Improved clearance during treatment of HPV-positive head 13. Stern PL, et al. (2012) Therapy of human papillomavirus-related disease. Vaccine and neck cancer through mTOR inhibition. Neoplasia 15(6):620–630. 30(Suppl 5):F71–F82. 43. Grabiner BC, et al. (2014) A diverse array of cancer-associated MTOR mutations are

14. Bertout JA, Patel SA, Simon MC (2008) The impact of O2 availability on human cancer. hyperactivating and can predict rapamycin sensitivity. Cancer Discov 4(5):554–563. Nat Rev Cancer 8(12):967–975. 44. Fruman DA, Rommel C (2014) PI3K and cancer: Lessons, challenges and opportunities. 15. Höckel M, Vaupel P (2001) Tumor hypoxia: Definitions and current clinical, biologic, Nat Rev Drug Discov 13(2):140–156. and molecular aspects. J Natl Cancer Inst 93(4):266–276. 45. Chiarini F, Evangelisti C, McCubrey JA, Martelli AM (2015) Current treatment strate- 16. Vaupel P, Mayer A (2007) Hypoxia in cancer: Significance and impact on clinical gies for inhibiting mTOR in cancer. Trends Pharmacol Sci 36(2):124–135. outcome. Cancer Metastasis Rev 26(2):225–239. 46. Lim HJ, Crowe P, Yang JL (2015) Current clinical regulation of PI3K/PTEN/Akt/mTOR 17. Vaupel P (2009) Pathophysiology of solid tumors. The Impact of Tumor Biology on signalling in treatment of human cancer. J Cancer Res Clin Oncol 141(4):671–689. Cancer Treatment and Multidisciplinary Strategies, eds Molls M, Vaupel P, Nieder C, 47. Leontieva OV, Blagosklonny MV (2010) DNA-damaging agents and p53 do not cause Anscher MS (Springer, Berlin, Heidelburg), pp 51–92. senescence in quiescent cells, while consecutive re-activation of mTOR is associated 18. Bachtiary B, et al. (2003) Overexpression of hypoxia-inducible factor 1alpha indicates with conversion to senescence. Aging (Albany NY) 2(12):924–935. diminished response to radiotherapy and unfavorable prognosis in patients receiving 48. Herranz N, et al. (2015) mTOR regulates MAPKAPK2 translation to control the se- radical radiotherapy for cervical cancer. Clin Cancer Res 9(6):2234–2240. nescence-associated secretory phenotype. Nat Cell Biol 17(9):1205–1217. 19. Birner P, et al. (2000) Overexpression of hypoxia-inducible factor 1alpha is a marker for an 49. Laberge RM, et al. (2015) MTOR regulates the pro-tumorigenic senescence-associated unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 60(17):4693–4696. secretory phenotype by promoting IL1A translation. Nat Cell Biol 17(8):1049–1061. 20. Sørensen BS, et al. (2013) Radiosensitivity and effect of hypoxia in HPV-positive head 50. Guba M, et al. (2002) Rapamycin inhibits primary and metastatic tumor growth by an- and neck cancer cells. Radiother Oncol 108(3):500–505. tiangiogenesis: Involvement of vascular endothelial growth factor. Nat Med 8(2):128–135. 21. Vaupel P, Höckel M, Mayer A (2007) Detection and characterization of tumor hypoxia 51. Hengstermann A, Linares LK, Ciechanover A, Whitaker NJ, Scheffner M (2001) Com-

using pO2 histography. Antioxid Redox Signal 9(8):1221–1235. plete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in 22. Semenza GL (2014) Oxygen sensing, hypoxia-inducible factors, and disease patho- cervical cancer cells. Proc Natl Acad Sci USA 98(3):1218–1223. physiology. Annu Rev Pathol 9:47–71. 52. Koumenis C, et al. (2001) Regulation of p53 by hypoxia: Dissociation of transcriptional re- 23. Chai TF, et al. (2011) Hypoxia-inducible factor independent down-regulation of thi- pression and apoptosis from p53-dependent transactivation. Mol Cell Biol 21(4):1297–1310. oredoxin-interacting protein in hypoxia. FEBS Lett 585(3):492–498. 53. Achison M, Hupp TR (2003) Hypoxia attenuates the p53 response to cellular damage. 24. Li Y, et al. (2013) HIF- and non–HIF-regulated hypoxic responses require the estrogen- Oncogene 22(22):3431–3440. related receptor in Drosophila melanogaster. PLoS Genet 9(1):e1003230. 54. Meugnier E, Rome S, Vidal H (2007) Regulation of gene expression by glucose. Curr 25. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM (1990) The E6 on- Opin Clin Nutr Metab Care 10(4):518–522. coprotein encoded by human papillomavirus types 16 and 18 promotes the degra- 55. Havula E, Hietakangas V (2012) Glucose sensing by ChREBP/MondoA-Mlx transcrip- dation of p53. Cell 63(6):1129–1136. tion factors. Semin Cell Dev Biol 23(6):640–647. 26. Vassilev LT, et al. (2004) In vivo activation of the p53 pathway by small-molecule 56. Leitz J, et al. (2014) Oncogenic human papillomaviruses activate the tumor-associated antagonists of MDM2. Science 303(5659):844–848. lens epithelial-derived growth factor (LEDGF) gene. PLoS Pathog 10(3):e1003957. 27. Leontieva OV, Blagosklonny MV (2012) Hypoxia and gerosuppression: The mTOR saga 57. Korotchkina LG, et al. (2010) The choice between p53-induced senescence and qui- continues. Cell Cycle 11(21):3926–3931. escence is determined in part by the mTOR pathway. Aging (Albany NY) 2(6):344–352. 28. Leontieva OV, Demidenko ZN, Gudkov AV, Blagosklonny MV (2011) Elimination of 58. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real- proliferating cells unmasks the shift from senescence to quiescence caused by rapa- time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. mycin. PLoS One 6(10):e26126. 59. Schmitz M, et al. (2012) Loss of gene function as a consequence of human papillo- 29. Leontieva OV, et al. (2012) Hypoxia suppresses conversion from proliferative arrest to mavirus DNA integration. Int J Cancer 131(5):E593–E602. cellular senescence. Proc Natl Acad Sci USA 109(33):13314–13318. 60. Tóth ZE, Mezey E (2007) Simultaneous visualization of multiple antigens with tyr- 30. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibits amide signal amplification using antibodies from the same species. J Histochem S6Ks and 4E-BP1 to mediate cell type-specific repression of mRNA translation. Proc Cytochem 55(6):545–554. Natl Acad Sci USA 105(45):17414–17419. 61. Hopman AH, Ramaekers FC, Speel EJ (1998) Rapid synthesis of biotin-, digoxigenin-, 31. Kang SA, et al. (2013) mTORC1 phosphorylation sites encode their sensitivity to trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ starvation and rapamycin. Science 341(6144):1236566. hybridization using CARD amplification. J Histochem Cytochem 46(6):771–777.

E998 | www.pnas.org/cgi/doi/10.1073/pnas.1615758114 Hoppe-Seyler et al. Downloaded by guest on October 3, 2021