c-MYC-dependent transcriptional inhibition of autophagy is implicated in Cisplatin sensitivity in HPV-positive Head and Neck Cancer

doi:10.21203/rs.3.rs-2468300/v1

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c-MYC-dependent transcriptional inhibition of autophagy is implicated in Cisplatin sensitivity in HPV-positive Head and Neck Cancer

https://doi.org/10.21203/rs.3.rs-2468300/v1

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published 04 Nov, 2023

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Autophagy is important for removal, degradation and recycling of damaged organelles, proteins, and lipids through the degradative action of lysosomes. In addition to its catabolic function, autophagy is important in cancer and viral-mediated tumorigenesis, including Human Papillomavirus (HPV) positive cancers. HPV infection is a major risk factor in a subset of Head and Neck Cancer (HNC), for which no targeted therapies are currently available.

Herein, we assessed autophagy function in HPV-positive HNC. We showed that HPV-positive HNC cells presented a transcriptional and functional impairment of autophagic process compared to HPV-negative cells, which was reactivated by knocking-down HPV E6/E7 oncoproteins, the drivers of cellular transformation. We found that the oncoprotein c-MYC was stabilized and triggered in HPV-positive cell lines. This resulted in the reduced binding of the MiT/TFE transcription factors to their autophagy targets due to c-MYC competition. Thus, knock-down of c-MYC induced the upregulation of autophagic and lysosomal genes in HPV-positive HNC cells, as well as the increase of autophagic markers at the protein level.

Moreover, HPV oncoprotein E7 upregulated the expression of the phosphatase inhibitor CIP2A, accounting for c-MYC upregulation and stability in HPV + HNC cells. CIP2A mRNA expression negatively correlated with autophagy gene expression in tumor tissues from HNC patients, showing, for the first time, its implication in a transcriptional autophagic context. Both CIP2A and c-MYC knock-down, as well as pharmacological downregulation of c-MYC, resulted in increased resistance to Cisplatin treatment.

Our results not only show a novel way by which HPV oncoproteins manipulate the host machinery, but also provide more insights on the role of autophagy in chemoresistance, with possible implications for targeted HPV-positive HNC therapy.

Head and Neck cancer (HNC) comprises tumors originating in the epithelial cells of the mucosa of the upper aerial tract, including the oropharynx, the oral cavity, the larynx and the hypopharynx (1). Based upon its main risk factors, alcohol/tobacco consumption and Human Papillomavirus (HPV) infections, HNC can be subdivided into two subgroups: HPV-negative (HPV-) and HPV-positive (HPV+) (2). HPV-related HNC mostly occurs in the oropharyngeal subsite and has been increasing in the last decades, especially in men (3). Indeed, HPV + oropharyngeal cancers have one of the fastest rises in incidence in non-hispanic white men in the United States (4). Although the two HNC subtypes are different in both clinical and molecular aspects, they are still treated with the same therapies, including surgery, when possible, followed by radio- and chemo-therapy (5). A better knowledge of the molecular aspects that distinguish these two subgroups would be beneficial for the discovery of novel targets and biomarkers for targeted therapy.

HPV is a sexually transmitted double strand DNA virus of approximately 8000 bp (6). Over 200 HPV strains exist, subdivided into high-risk or low-risk for the capability to induce carcinogenesis (7). The main drivers of oncogenesis are the E6 and E7 oncoproteins: E6 interacts with many cellular proteins, including p53, which, upon interaction with the human ubiquitin-ligase E6AP, is sent to proteasomal degradation, leading to inhibition of apoptosis (8). E7 mainly induces retinoblastoma protein (pRb) inactivation, leading to E2F1 release, an important transcription factor family involved in cell cycle regulation, resulting in increased proliferation (9, 10).

Another target gene activated by E2F1 is the Cellular Inhibitor of PP2A (Protein Phosphatase 2A) (CIP2A) (11, 12). CIP2A sequesters PP2A B56 regulatory subunit inhibiting its activity and, as a consequence, increases the half-life of the oncogene c-MYC by inhibiting its S62 dephosphorylation by PP2A (13).

Autophagy is a self-consumption intracellular process important for catabolism and homeostasis (14). Macroautophagy, from here after referred to as autophagy, consists in the formation of a double membrane organelle, the autophagosome (15), on which the light chain protein 3 (LC3) is loaded on upon maturation to a lipidated form through a ubiquitin-like system (16, 17). LC3 is extensively used to monitor the autophagic flux (18). The autophagosome, containing a cargo of proteins and damaged organelles, is degraded upon fusion with lysosomes, degradative organelles consisting of digestive enzymes and an acidic environment (19). Autophagy is important for the recycling of amino acids, for the degradation of long-lived proteins, and the recycling of damaged organelles, including mitochondria (20). Autophagy and lysosomal biogenesis can be transcriptionally regulated by different transcription factors, in particular the microphthalmia/transcription factor E (MiT/TFE) family (21, 22). This family includes microphthalmia-associated transcription factor (MITF), transcription factor E3 (TFE3), transcription factor EB (TFEB) and transcription factor EC (TFEC) proteins, belonging to the larger family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors (that include also c-MYC), that can recognize a specific motif (GTCACGTGAC) known as the coordinated lysosomal expression and regulation (CLEAR) (22). Specifically, TFEB and TFE3 activities are controlled by phosphorylation: mechanistic target of rapamycin complex 1 (mTORC1) inhibition dephosphorylates TFEB/TFE3 inducing nuclear localization and activation of lysosome/autophagy gene expression (23).

The role of autophagy in cancer is still not well understood. Many works suggest that autophagy inhibition drives tumorigenesis (24–26), however other studies suggest that autophagy induction can insure cancer cell survival in advanced solid tumors (27, 28). Autophagy is considered as the double-edged sword in cancer (29).

The role of HPV in autophagy regulation is emerging, and in particular E6 and E7 oncoproteins impinge this pathway at different steps (30). E6 has been shown to inhibit late steps of autophagy in primary keratinocytes (31), while E7 has been observed to inhibit autophagy by AMBRA1 degradation (32).

In this study we focused on autophagy gene expression regulation mediated by E6/E7 in HNC and how c-MYC affects this process. We also tested the implication of PP2A activation or c-MYC downregulation in combination with Cisplatin in HPV + HNC to assess their role in chemo-sensitivity.

1. Autophagy is structurally and functionally impaired in HPV + HNC cell lines

To assess the differences in autophagy activation between HPV + and HPV- HNC cells, we examined the number of autophagic vacuoles (AVs) (which include autophagosomes, autolysosomes and amphisomes) by electron microscopy (EM) in 2 HPV- and 2 HPV + HNC cell lines (Fig. 1A). HPV + HNC cell lines showed a reduced number and size of AVs compared to HPV- ones, suggesting a possible inhibition of the pathway (Fig. 1B, S1A).

We then assessed the levels of autophagy markers in HNC cell lines at basal conditions. HPV + HNC cell lines showed lower levels of the lipidated form of LC3 (LC3-II) compared to HPV- HNC cell lines (Fig. 1C, 1D), while other autophagy markers like Beclin or p62 were only respectively slightly or not affected (Figure S1B, S1C). We also measured the activation of Akt and mTORC, described to inhibit autophagy (33), by detecting Akt and p70 S6 kinase (S6K) phosphorylation by western blot in HNC cell lines (Figure S1D, S1E). While Akt phosphorlylation at S473 seems not to be related to HPV status, p70 (S6K) phosphorylation can be detected in the UD-SCC-2 and UPCI-SCC-90 HPV + HNC cell lines, suggesting that mTORC activity could promote autophagy inhibition in some HPV + HNC cell lines.

To functionally characterize autophagy status, we monitored the autophagic flux by using a tandem tagged LC3-GFP-RFP fusion protein (34). GFP is quenched in acidic pH such that only red dots can be observed in functional autolysosomes, while in neutral pH red and green colocalized dots can be detected. The HPV- UM-SCC-19 and the HPV + UD-SCC-2 HNC cell lines transduced with the lentiviral LC3-GFP-RFP vector were treated with the late stage autophagic inhibitor Bafilomycin A1, the mTORC1 inhibitor Torin-1, or the vehicle (DMSO) and observed by confocal microscopy (Fig. 1E). Quantification of colocalized GFP and RFP dots showed a significant increase upon autophagy inhibition and a significant decrease upon autophagy activation with respect to the vehicle only in the HPV- HNC cell lines, as expected in cells in which autophagy is functional (Fig. 1F, left). However, the levels of colocalized dots in the HPV + HNC cell lines do not vary, or increase, with respect to the vehicle, upon pharmacological induction or inhibition of autophagy (Fig. 1F, right). Taken together, these results suggest a global impairment in autophagic functions in HPV + HNC cell lines.

2. Hpv16 Oncoproteins Transcriptionally Inhibit Autophagy Gene Expression

To test whether HPV16 oncoproteins are involved in autophagy inhibition in HPV + HNC cells, we knocked down E6/E7 by RNAi in UD-SCC-2 cells. We then stained Lysosome-associated membrane protein 2 (LAMP2) (lysosomal) and LC3 (autophagosomal) proteins, revealing an increase in colocalization upon reduction of E6/E7, suggesting a reactivation of the pathway (Fig. 2A, 2B). This result was confirmed also by a significant increase in the LC3II/LC3I ratio upon siE6/E7 (Fig. 2C, 2D). Interestingly, downregulation of E6 and E7 was able to induce the mRNA upregulation of some autophagic and lysosomal genes (Fig. 2E).

To investigate the role of E6 and E7 in mRNA regulation of autophagy and lysosomal biogenesis, we transduced primary human keratinocytes (PHK) with HPV16 E6 and E7 (Fig. 2F, S2A), followed by RNA sequencing. Gene ontology analysis on genes downregulated by E6/E7 indicated genes belonging to lysosomal and vacuolar cellular compartments as some of the most relevant genes affected by the two oncoproteins (Fig. 2G), in addition, the pathway analysis highlighted lysosome and autophagy pathway among the top regulated hits (Fig. 2H). Moreover, only HPV16 E7 alone, and not E6, seems to induce mRNA downregulation of autophagy and lysosomal biogenesis (Figure S2D, S2E). These results were also confirmed by qPCR analysis (Figure S2B, S2C).

Next, we wanted to confirm these results also in HNC cell lines by monitoring the mRNA expression of autophagy and lysosomal genes in two HPV- (UM-SCC-4, UM-SCC-19) and two HPV+ (UD-SCC-2, UPCI-SCC-152) HNC cell lines at basal conditions. Figure 2I shows that many autophagy (the LC3 gene MAP1LC3B, the p62 gene SQSTM1, BECN1, GABARAP, GABARAPL1) and, more strongly, lysosomal (NEU1, CTSA, CTSB, CTSD, ATP6V0E1, LAMP1, LAMP2) genes are differentially expressed between HPV- and HPV + cell lines. These data suggest that HPV16 oncoproteins, and in particular E7, transcriptionally downregulate autophagy and lysosomal biogenesis.

3. c-MYC competes with TFEB and TFE3 for the binding to autophagy and lysosomal genes in HPV + HNC cells

As transcriptional repression of autophagy and lysosomal biogenesis occurred in HPV + cells, we investigated the molecular mechanism underlying this process. We evaluated the mRNA and protein expression (Figure S3A-C, Fig. 3A) of the most important transcription factors involved in lysosomal biogenesis: TFEB and TFE3, as well as c-MYC, recently found to repress autophagy (35). Western blots performed on HNC cell lines did not show any significant difference in the expression of these genes between HPV- and HPV + cell lines (Fig. 3A).

Thus, we focused on the differential activation of the transcription factors, and on their binding to the promoters of autophagy genes. We performed chromatin immunoprecipitation of TFEB, TFE3, and c-MYC in the UM-SCC-4 and UD-SCC-2 cell lines, followed by qPCR analysis of autophagy and lysosomal genes. We found that the binding of c-MYC to the promoter of LC3 gene (MAP1LC3B) was significantly higher in the HPV + cell line (UD-SCC-2) compared to the HPV- cell line (UM-SCC-4), and this corresponded to a significant decrease in the binding of TFEB and TFE3 to the same promoter (Fig. 3B). The same result was obtained also for GLA and COLN1 promoters (Fig. 3C, 3D), while no significant differences could be appreciated for the promoter of p62 gene (SQSTM1) (Fig. 3E). This is in line with Annunziata et al. (35) study, which showed that c-MYC competes with TFE3 and TFEB, repressing lysosomal and autophagic transcription. Our data suggest that HPV + HNC cells present a more active c-MYC compared to HPV- cells.

4. C-myc Stability Increases In Hpv + hnc Cells, Affecting The Autophagy Pathway

To assess whether c-MYC is involved in the inhibition of autophagy, we performed c-MYC knock-down in HPV + HNC cell lines. c-MYC downregulation affected both protein expression of LC3 and the mRNA levels of different autophagy genes (Figs. 4A, 4B, S4A, S4B, S4D). Moreover, c-MYC overexpression UD-SCC-2 and UPCI-SCC-152 cell lines was able to affect LC3 protein levels and to induce the downregulation of autophagy genes, confirming the involvement of c-MYC in autophagy repression (Figs. 4C, S4C, 4D).

Since no significant difference in the mRNA expression of MYC between HPV + and HPV- HNC cell lines was detected (Figure S3C), we evaluated the stability of c-MYC protein by treating HNC cell lines with cycloheximide (CHX), a widely used inhibitor of protein synthesis, at different time points. Since c-MYC is very unstable, we used 5 minutes as the first time point and 90 minutes as the last. Western blot analysis revealed a higher stability of c-MYC in HPV + with respect to HPV- HNC cell lines (Fig. 4E, S4E, S4F). We calculated the half-life of c-MYC in 3 HPV- and 3 HPV + HNC cell lines (Fig. 4F) and we observed a significant higher half-life in the HPV + HNC cell lines, revealing a higher stability of c-MYC in these cell lines (Fig. 4G). Moreover, E6/E7 silencing in the HPV + UD-SCC-2 cell line induced the downregulation of c-MYC protein level (Figure S4G), suggesting that the stability of c-MYC relies on E6/E7 oncoproteins.

Since c-MYC stability is affected by its phosphorylation, we tried to assess whether the phosphatase inhibitor was responsible for c-MYC stabilization in the UD-SCC-2 cell line. Silencing of CIP2A by shRNA induced a strong downregulation of c-MYC protein levels (Fig. 4H), which was specifically imputable to the reduction of c-MYC half-life (Fig. 4I). These results confirmed our hypothesis that, also in our system, c-MYC protein stability is regulated by its phosphorylation controlled by CIP2A expression.

5. Cip2a Inhibits Autophagy And Lysosomal Biogenesis In Hnc

As CIP2A affected c-MYC stability, we investigated the role of CIP2A mRNA expression in HNC tumors. We took advantage of the Pancancer Dataset of Head and Neck cancer tumors from the Cancer Genome Atlas (TCGA) and assessed the correlation between CIP2A mRNA and autophagy or lysosomal genes expression. We found that many genes involved in autophagy and lysosomal biogenesis inversely correlated with CIP2A expression (Fig. 5A, S5A, S5B, S5C). We then took the top 100 genes that inversely correlated with CIP2A expression and performed a gene ontology analysis, looking at cellular components, biological processes and pathways enriched (Figs. 5B, 5C, 5D). As shown in Fig. 5B-D, among the most enriched hits we found lysosome related genes, macroautophagy, mitophagy and selective autophagy.

We then decided to assess whether CIP2A expression affects autophagy in HPV + HNC cell lines. Upon CIP2A silencing in HPV + HNC cell lines we showed the upregulation of LC3-II (Fig. 5E and 5F), and also the increased mRNA LC3 gene expression (Fig. 5G). Moreover, the overexpression of CIP2A was able to reduce the colocalization of LC3 with LAMP2 (Figs. 5H, 5I). Taken together these results evidence a novel and important role of CIP2A expression in autophagy gene expression in HNC.

6. Hpv16 E6/e7 Induce Cip2a Overexpression In Hnc

After demonstrating the importance of CIP2A in c-MYC stability and autophagy in HNC, we evaluated the expression of CIP2A in HPV + vs HPV- HNC cell lines. Upon assessing mRNA expression of CIP2A in the Pancancer dataset, we observed significant higher expression levels in HPV + compared to HPV- HNC samples. (Fig. 6A). Interestingly, the same result was confirmed also in primary tumors obtained from IEO hospital (Fig. 6B), and in HNC cell lines (Fig. 6C).

We then evaluated the possible regulation of HPV16 E6/E7 on CIP2A expression, by transducing PHK with E6/E7 (Fig. 6D). Western blot analysis showed the upregulation of CIP2A in the presence of E6 and E7 proteins, confirmed by higher mRNA levels of CIP2A (Figure S6D).

To strengthen our observations, we checked CIP2A protein levels in HPV + and HPV- HNC cell lines, confirming significant higher levels of CIP2A in the HPV + subgroup (Fig. 6E, 6F), and that this upregulation can be significantly reverted upon knock-down of E6/E7 (Fig. 6G, 6H).

These results are in line with published literature showing an overexpression of CIP2A by high risk HPV due to E7 inhibition of pRb in other systems (36–38). Along the same line, we also observed the overexpression of E2F1 (regulated by pRb) both in TCGA cases and in HNC cell lines (Figure S6A, S6C). Moreover, gene set enrichment analysis (GSEA) performed on RNAseq data from PHK transduced with E6/E7 showed a significant enrichment in E2F1 pathways and E2F targets, as well as MYC upregulation, as expected (S6E, S6F, S6G). The same analysis did not show any significant enrichment in mTORC signaling (Figure S6H, S6I). All these data suggest that HPV indirectly controls CIP2A expression activating E2F1 signaling upon pRb inhibition.

7. Cip2a And C-myc Downregulation Confer Resistance To Cisplatin In Hpv + hnc Cell Lines

Autophagy has a fundamental role in resistance to chemotherapy (39–41). Recent findings suggest that inhibition of autophagy sensitizes cells to Cisplatin treatment (42, 43). It has been described that HPV + HNC cells are more sensitive to Cisplatin than HPV- cells (44). To understand the role of c-MYC and CIP2A inhibition in chemosensitivity, we evaluated Cisplatin IC50 in the HPV + UD-SCC-2 cell line upon knock-down of c-MYC or CIP2A. Figure 7A and 7B show that the downregulation of both c-MYC and CIP2A induced resistance to Cisplatin, with almost a doubling of the IC50 values in UD-SCC-2 cells.

To confirm these results, we downregulated c-MYC pharmacologically, by treating UD-SCC-2 and UPCI-SCC-152 cells with the Brd4 inhibitor JQ1, already known to activate autophagy (45, 46). We also tested the effects of the PP2A activator DT-061, to abrogate the impact of CIP2A overexpression on c-MYC (47). Both treatments were able to downregulate c-MYC levels (Figure S7C, S7D). JQ1 efficiently downregulated mRNA MYC expression and to upregulate autophagy in UD-SCC-2 and UPCI-SCC-152 cell lines (S7A, S7B). We then assessed the IC50 of Cisplatin in the presence of 1µM JQ1, 1µM DT-061 or vehicle (DMSO). Figure 7C and 7D showed an increased resistance to Cisplatin in comparison to the control in UD-SCC-2 cells. These results were confirmed also in the UPCI-SCC-152 cell line (Fig. 7E, 7F).

Taken together these results suggest the importance of c-MYC activity in chemosensitivity in HPV + HNC cells.

Autophagy regulation and its role in HPV-related carcinogenesis are far from being well characterized. However, emerging evidence showed the role of HPV16 E6 and E7 oncoproteins as inhibitors of autophagy in various cellular models (30). Different mechanisms have been proposed, but only few studies have been conducted in HNC cells (32, 48, 49). We showed for the first time by electron microscopy that autophagic structures are impaired in HPV + cell lines compared to HPV- ones. The phenotypic reduction of these structures was due to a wider transcriptional program, in which autophagy and lysosomal biogenesis were inhibited by viral oncoproteins. Specifically, we were able to understand that HPV16 E7 alone was sufficient to repress the transcription of autophagy and lysosomal genes.

HPV oncoproteins E6 and E7 have been both described to interact with c-MYC, to upregulate its transcriptional activity, and only E6 to also increase its phosphorylated fraction (50, 51). In this study, we showed a higher stability of c-MYC, leading to a higher binding to autophagy genes promoters, inhibiting TFEB and TFE3 activity in HPV + HNC cell lines. The concept of c-MYC preventing TFE3 and TFEB to bind to autophagy and lysosomal genes was previously showed by Annunziata et al., and shed light on how c-MYC can inhibit transcription of autophagy genes (35). This is also in line with some findings in which promotion of c-MYC dephosphorylation and degradation upregulates autophagy levels (52).

We showed that the viral oncoproteins enhance c-MYC half-life increasing its phosphorylated form. As described above, CIP2A inhibits the effects of PP2A on S62 of c-MYC, important for c-MYC stability and phosphorylation (13). Here we showed how CIP2A is important in c-MYC stabilization in HPV + HNC, and its involvement in autophagy inhibition. CIP2A is known to regulate autophagy (53), however upon interrogating the TCGA we assessed for the first time a negative correlation between its expression and autophagy/lysosomal gene expression in HNC patients. Puustinen et al. described that CIP2A inhibits autophagy in a mTORC dependent manner (54), however our results on mTORC involvement are not consistent. Moreover, mTORC inhibition by Torin-1 did not induce a reduction in LC3 colocalized dots, and RNA-seq on PHK did not show any significant enrichment in mTORC pathway. More studies are needed to better clarify the contribution of this pathway in a CIP2A overexpressed environment.

We demonstrated that the oncoviral proteins E6/E7 upregulated CIP2A mRNA and protein levels in HNC cells and in PHK, in line with Zhang et al. (37). CIP2A, overexpressed in HPV + HNC cell lines, in HPV + cases from the TCGA, and also in fresh primary tumors from HNC patients, could be used as a potential marker for HPV + tumors. CIP2A was probably due to E2F1 overactivation, that we also showed in different systems (Figure S6A, S6C, S6F, S6G). E7, mediating pRb impairment, induces the upregulation of E2F1, a known regulator of CIP2A (36–38).

In HPV- HNC cells E2F activity is tightly regulated, CIP2A is not overexpressed and c-MYC has a short half-life. The expression of autophagy genes is controlled by TFE3 and TFEB and autophagy is functional (Fig. 8A). However, HPV + HNC cells express high levels of CIP2A due to overactivation of E2F as a consequence of E7-mediated degradation of pRb. This leads to high activity and stability of c-MYC that binds to autophagy and lysosomal gene promoters, resulting in the inhibition of autophagy pathway (Fig. 8B).

The relevance of these findings was extended to resistance to chemotherapy. As mentioned above, HPV + HNC cell lines are known to be more sensitive to Cisplatin (44). The notion that autophagy could be important for resistance to chemotherapy (32, 55) was confirmed in this study, in which we showed that downregulation of c-MYC, responsible for autophagy inhibition, resulted in increased resistance to cisplatin. The link between HPV, c-MYC and autophagy could give rise to novel approaches to treat HNC patients. Moreover, these results could pave the way to explore how autophagy can be manipulated, to propose more tailored and effective treatments to patients, reducing side effects and toxicity due to high doses of chemotherapy, which are often detrimental for patients.

Cell culture

HNC cell lines used in this study (56), were described and cited in previous studies (57, 58), while the laryngeal UM-SCC-17A cell line derives from a laryngeal primary carcinoma of a 48-years old female patient (Millipore). Cells were grown in Dulbecco’s modified Eagle’s medium containing stable glutamine (Euroclone) supplemented with antibiotics, 10% FBS (Fisher Scientific) and non-essential amino-acids (NEAA) and Plasmocin (Invivogen) to prevent mycoplasma contamination. Every 6 months all cell lines were authenticated by short tandem repeat profiling and tested for mycoplasma contamination. Skin biopsies from both male and female donors were collected and anonymized via standardized operative procedures approved by European Institute of Oncology Ethical Board. Informed consent was obtained from all patients. Adult human epidermal keratinocytes (HKs) were prepared and maintained as previously described (31, 59). Primary cultures of the isolated cells were then maintained in Keratinocyte Serum-Free Medium (KSFM; Gibco) containing bovine pituitary extract (BPE, 30 µg/mL; Gibco) and epidermal growth factor (EGF, 0.2 ng/mL; Gibco). Cells from passages 2–5 were used for the experiments. All cells were cultured at 37°C in a 5% CO₂ buffered incubator.

Tissue Samples

Fresh tumors and adjacent normal tissue samples from both male and female HNC patients were separately collected upon surgery under IEO Biobank approval and preserved at -80°C. Samples were then snap-frozen in liquid nitrogen and processed for RNA extraction using AllPrep DNA/RNA/protein kit (Qiagen) following manufacturers’ instruction. HPV16 presence was confirmed by qPCR (Figure S6B).

Transduction, Transfection And Plasmids

pLXSN HPV16 E6/E7, pLXSN HPV16 E6, pLXSN HPV16 E7 and empty vector were previously described (31). pLKO.1 PURO shluc and pcDNA3-MYC were a gift from B. Amati (IEO). pcDNA3-CIP2A-v5, pLKO.1 PURO shCIP2A#1, pLKO.1 PURO shCIP2A#1 and pLKO.1 PURO empty vector were a gift from S.Minucci (IEO). pLKO.1 PURO empty vector was subcloned as described in Ravasio et al. (60) using oligos described in Supplementary Information section to generate pLKO.1 PURO shMYC#1 and pLKO.1 PURO shMYC#2 plasmids. pLKO.1 PURO shCIP2A#1 and pLKO.1 PURO shCIP2A#2 targeting sequences are listed in Supplementary Information section. pLenti CMV GFP-LC3-RFP was a generous gift from J. Yue (University of Hong Kong, Hong Kong, China). Retroviral and lentiviral transductions were performed as described in Mattoscio et al. (31). HNC cell lines were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) to overexpress pcDNA3 MYC, pcDNA3 CIP2A-v5 plasmids, or the empty vector. siRNAs listed in supplementary information section were transfected using RNAiMAX reagent (Thermo Fisher Scientific), following manufacturers’ instructions.

Electron Microscopy

HNC cells were plated in growth medium at a density of 10⁵ cells/well in 6-well plates and grown for 24 hours. Cells were fixed in 2.5% glutaraldehyde (Sigma) solution for 1 hour at room temperature. After fixation cells were rinsed with 0.1M sodium cacodylate (Sigma) buffer (pH 7.4), three times for 5 minutes at room temperature. Cells were post-fixed in a solution containing 1% OsO₄ (Sigma) 1.5% K₄Fe(CN)₆ (Sigma), and 0.1M sodium cacodylate for 1 hour at 4ºC protected from light. After fixation samples were washed 3 times for 5 minutes with 0.1M sodium cacodylate buffer and washed 5 times with distilled water. Samples were then stained with 0.5% uranyl acetate at 4 ºC overnight, protected from light. After 5 washing steps with distilled water, samples were dehydrated using the following increasing concentration of ethanol (5 minutes per step): 30%, 50%, 70%, 80%, 90% and 96%. Then, samples were incubated 3 times for 5 minutes in 100% ethanol. After dehydration, samples were covered with a mixture of ethanol and epoxy resin (1:1) and left for 2 hours on a shaker at room temperature. After 2 changes of pure epoxy resin (1 hour per change) at room temperature, samples were finally embedded in fresh epoxy resin and polymerized overnight at 45 ºC and then incubated at 60 ºC for 24 hours.

Ultrathin sectioning (70nm) was performed on a Leica EM UC6 ultramicrotome. Sections were picked up and positioned on a 300 mesh squares grid. Thin sections were contrasted with 2% aqueous uranyl acetate for 5 minutes, followed by 3 washes in filtered distilled water (1 minute each). Subsequently, grids were placed in a drop of Sato lead stain and incubated for 2 minutes, rinsed 3 times with pure water, and dried with Whatman filter paper. Images were collected using a FEI Tecnai-12 transmission electron microscope.

Autophagic structures, termed autophagic vacuoles (AVs), which collectively referred to autophagosomes, amphisomes, and autolysosomes were quantified using Image J software. Multivesicular bodies, endosomes, and phagophores were not included in the analysis. Quantification was performed in terms of number of AVs per cellular profile and in area occupied by AVs.

Confocal Microscopy

Cells expressing LC3-GFP-RFP were seeded on coverslips and incubated for 24 hours at 37 ºC. cells were treated with 1µM Torin-1 (Selleckchem), 10nM Bafilomycin A1 (Sigma Aldrich) or vehicle for 24 hours, fixed with 4% PFA. Nuclei were stained with DAPI. Coverslips were then mounted and images were taken using a Leica SP8 AOBS (Leica microsystems) confocal microscopy using 63X oil objective and acquired with Leica Confocal Software. Quantification of dots number and colocalization were measured adapting the previously reported Red and Green Puncta Colocalization plugin and macro of ImageJ software (61).

Cells transfected with plasmids or siRNA were plated on glass coverslips were fixed with ice-cold methanol/acetone (1:1) for 2 minutes at -20°C, washed and blocked with 5% BSA for 30 minutes. Cells were then incubated with primary antibodies for 45 minutes at room temperature. The following antibodies (specified in supplementary information section) were used: anti-LC3 1:200, anti-LAMP2 1:100. Next, cells were incubated with Alexa Fluor (Life technologies) 488 or 647 fluorescent dye-conjugated secondary antibodies for 30 minutes at room temperature. Nuclei were counterstained with DAPI, mounted and analyzed by confocal microscopy Leica SP8 AOBS (Leica microsystems).

Cell Lysis And Western Blotting

Cells were lysed in a sodium dodecyl sulfate (SDS) lysis previously described (58). After lysis, an equal amount of protein for each sample was resuspended in denaturing sample loading buffer, separated on SDS-PAGE gel and immunoblotted with the indicated antibodies. The list of antibodies is reported in supplementary information section. Membranes were then incubated with the appropriate horseradish peroxidase (HRP) secondary antibodies and the signal was acquired with Chemidoc (Bio-Rad). Densitometric analysis of the intensity of the protein bands were preformed using ImageLab software (Bio-Rad).

Rt-qpcr

RNA was extracted from cells with the Quick-RNA MiniPrep kit (Zymo research). cDNA was generated by reverse transcription with Lunascript RT Supermix (New England Biolabs). Relative levels of specific mRNAs were determined with the Luna Universal qPCR master mix (New England Biolabs). All PCR reactions were performed with a QuantStudio 6 Fast Real-Time PCR system (Thermo Fisher Scientific). Neutral Ribosomal Phosphoprotein P0 (RPLP0) was used as a house-keeper gene for normalization. mRNA expression was calculated using the 2^−∆CT or 2^−∆∆CT methods. Primers used are listed in supplementary information section.

Chromatin Immunoprecipitation (ChIP)

10⁷ UD-SCC-2 and UPCI-SCC-152 cells treated with 250nM Torin-1 for 16 hours were fixed with 1% formaldehyde for 10 minutes at RT. 125µM were used to quench formaldehyde. After 3 PBS washes cells were harvested, centrifuged at 300 rcf and lysed using Lysis Buffer 1 (50mM Hepes, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) in the presence of protease inhibitors. After 10 minutes on ice cells were centrifuged at 300 rcf and resuspended in Lysis Buffer 2 (10mM Tris-Hcl pH8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) in the presence of protease inhibitors and rocked gently for 10 minutes at RT. After centrifugation (300 rcf) nuclei were lysed in Lysis Buffer 3 (10mM Tris-Hcl pH8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-laureylsarcosine). Chromatin was sheared by sonication, to obtain a bulk size of 200-500bp. Chromatin was then quantified using DC assay (Biorad). 500µg chromatin were incubated overnight at 4°C with Protein G Dynabeads (Invitrogen) coupled with 10µg of antibody. After immunoprecipitation, beads were recovered using a magnet and washed; chromatin was eluted using TE containing 2% SDS and cross-links reverted overnight at 65°C. DNA was purified with QIAquick columns (Qiagen). ChIPs were performed in triplicate for antibodies and in duplicate for IgGs. Antibodies used are listed in Supplementary Information section. 2 µL of the immunoprecipitated material or the purified input were used in qPCR reactions using the Luna Universal qPCR master mix (New England Biolabs) with primers listed in Supplementary Information section. All PCR reactions were performed with a QuantStudio 6 Fast Real-Time PCR system (Thermo Fisher Scientific). The values shown in the graphs are expressed as a percentage of the total input DNA.

Cell Viability Assay

UD-SCC-2 and UPCI-SCC-152 cell lines were seeded into 96-well white plates (2000 cells/well) and 24 h later were treated with 9 different concentrations of Cisplatin for 72 h and vehicle, alone or in combination with 1µM JQ1 (Medchem Express) or 1uM DT-061 (Medchem Express). UD-SCC-2 cell lines transduced with shluc, shMYC#1, shMYC#2, shCIP2A#1, shCIP2A#2 were seeded into 96-well white plates (2000 cells/well) and 24 h later were treated with 9 different concentrations of Cisplatin (Teva) for 72. Cell viability was then assessed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) following manufacturer instructions and luminescence was measured using a Glomax Discover plates reader (Promega).

RNA-seq data analysis

RNA form PHK transduced with E6, E7, E6/E7 or the empty vector was extracted from cells with the Quick-RNA MiniPrep kit according to the manufacturer’s instructions (Zymo Research). RNA quality and integrity were assessed with an Agilent 2100 bioanalyzer using an RNA 6000 Nano Kit (Agilent Technologies). mRNA-Seq library was prepared from 500 ng of total RNA with the TruSeq Stranded Total RNA (Illumina) and sequenced with Novaseq 6000 (Illumina) with a read length of 50 bp (paired-end). FastX-toolkits and FastQC tools were used to filter reads and control the quality of all raw data; TopHat was used for the alignment of reads to the reference genome. Alignment was performed using HTS-flow (62). A paired differential expression analysis was performed using the Bioconductor DESeq2 R package (63). Genes were identified as DEGs when false discovery rate (FDR) ≤ 0.05. Gene Ontology (GO) enrichment analyses were performed using SRplot tool (64, 65). Gene set enrichment analysis (GSEA) (66) was performed using all expressed genes ranked by signed P-value and 1,000 gene set permutations.

Statistical Analysis Of Tcga Data

RSEM normalized expression data was extracted from the TCGA PanCancer Atlas on the cBioPortal (67, 68). Patient samples with known HPV status were grouped as HPV + and HPV-. This resulted in 72 HPV + and 415 HPV-samples with data available for the HNC gene expression analysis. Violin plot comparisons of gene expression and statistical analysis were performed by https://www.bioinformatics.com.cn/en, a free online platform for data analysis and visualization.

Statistical analysis

Statistical analysis specified in figure legends were evaluated using GraphPad Prism v9.0 (Graphpad Software, Inc., San Diego, California, USA). P-value is represented as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Graphs were plotted using either using GraphPad Prism v9.0 (Graphpad Software, Inc., San Diego, California, USA) or https://www.bioinformatics.com.cn/en, a free online platform for data analysis and visualization. Figures and panels were designed using Affinity Designer. Some figures were created with Biorender.com.

Conflict of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Acknowledgements

We thank Andrea Raimondi and the ALEMBIC facility at San Raffele Hospital (Milan) for Electron Microscopy experiments. We are grateful to Bruno Amati (IEO, Milan) and Saverio Minucci (IEO, Milan) for plasmids. We thank Stefano Santaguida, Lavinia Ghiani, Claudia Miccolo and Sebastiano Peri for scientific discussion. Alessandro Medda was a PhD student within the European School of Molecular Medicine (SEMM). We are grateful to Adele Migliacci for the financial support.

Author Contribution Statement

A.M., Si. C. and Su. C. conceptualized the experiments. A.M., G.S. and M.C. performed the experiments. M.T. and M.A. obtained patients’ samples. A.M. and M.C. analyzed data. A.M. and O.C. performed bioinformatic analyses. A.M. prepared figures and panels. M.C. created the graphical abstract using biorender.com. A.M. wrote the original draft of the manuscript. Su.C. and Si.C. reviewed and edited the manuscript. Su. C. supervised the work. All authors read and approved the manuscript.

Ethics Statement

This study was carried out in accordance with the recommendations of IEO Biobank for translational medicine (B4MED). The protocol was approved by the IEO committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Funding Statement

This study was partially funded by Associazione Italiana per la Ricerca sul

Cancro (A.I.R.C.) to S. Chiocca (IG 2015 Id.16721), and the Italian Ministry of Health with Ricerca Corrente and 5 × 1000 funds. A. M is currently a recipient of the “Carlo Adamich” A.I.R.C. fellowship and S. Citro of a FUV (Fondazione Umberto Veronesi) fellowship.

Data Availability Statement

Sequencing data will be available upon acceptance of the manuscript.

All relevant data are within the paper and its Supporting Information files, further inquiries can be directed to the corresponding author.

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c-MYC-dependent transcriptional inhibition of autophagy is implicated in Cisplatin sensitivity in HPV-positive Head and Neck Cancer

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Journal Publication

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Abstract

Figures

Introduction

Results

1. Autophagy is structurally and functionally impaired in HPV + HNC cell lines

2. Hpv16 Oncoproteins Transcriptionally Inhibit Autophagy Gene Expression

4. C-myc Stability Increases In Hpv + hnc Cells, Affecting The Autophagy Pathway

5. Cip2a Inhibits Autophagy And Lysosomal Biogenesis In Hnc

6. Hpv16 E6/e7 Induce Cip2a Overexpression In Hnc

7. Cip2a And C-myc Downregulation Confer Resistance To Cisplatin In Hpv + hnc Cell Lines

Discussion

Material And Methods

Cell culture

Tissue Samples

Transduction, Transfection And Plasmids

Electron Microscopy

Confocal Microscopy

Cell Lysis And Western Blotting

Rt-qpcr

Chromatin Immunoprecipitation (ChIP)

Cell Viability Assay

RNA-seq data analysis

Statistical Analysis Of Tcga Data

Statistical analysis

Graphs And Figures

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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