Felix Hoppe‐Seyler

Effects of the antifungal agent ciclopirox in HPV‐positive cancer cells: Repression of viral E6/E7 oncogene expression and induction of senescence and apoptosis

The malignant growth of human papillomavirus (HPV)‐positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV‐positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV‐positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX‐induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53‐independent Caspase‐3/7 activation and induction of apoptosis. CPX also eliminates HPV‐positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV‐positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV‐inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.

Effects of Metformin on the virus/host cell crosstalk in human papillomavirus‐positive cancer cells

AbstractOncogenic types of human papillomaviruses (HPVs) are major human carcinogens. The viral E6/E7 oncogenes maintain the malignant growth of HPV‐positive cancer cells. Targeted E6/E7 inhibition results in efficient induction of cellular senescence, which could be exploited for therapeutic purposes. Here we show that viral E6/E7 expression is strongly downregulated by Metformin in HPV‐positive cervical cancer and head and neck cancer cells, both at the transcript and protein level. Metformin‐induced E6/E7 repression is glucose and PI3K‐dependent but—other than E6/E7 repression under hypoxia—AKT‐independent. Proteome analyses reveal that Metformin‐induced HPV oncogene repression is linked to the downregulation of cellular factors associated with E6/E7 expression in HPV‐positive cancer biopsies. Notably, despite efficient E6/E7 repression, Metformin induces only a reversible proliferative stop in HPV‐positive cancer cells and enables them to evade senescence. Metformin also efficiently blocks senescence induction in HPV‐positive cancer cells in response to targeted E6/E7 inhibition by RNA interference. Moreover, Metformin treatment enables HPV‐positive cancer cells to escape from chemotherapy‐induced senescence. These findings uncover profound effects of Metformin on the virus/host cell interactions and the phenotype of HPV‐positive cancer cells with implications for therapy‐induced senescence, for attempts to repurpose Metformin as an anticancer agent and for the development of E6/E7‐inhibitory therapeutic strategies.

Dickkopf‐1 expression is repressed by oncogenic human papillomaviruses (HPVs) and regulates the Cisplatin sensitivity of HPV‐positive cancer cells in a JNK‐dependent manner

AbstractOncogenic human papillomavirus (HPV) types control the phenotype of cervical cancer cells through the sustained expression of the viral E6/E7 oncogenes. Here, we show that they strongly restrain expression of the putative tumor suppressor protein Dkk1 (Dickkopf‐1) in HPV‐positive cervical cancer cells through the restriction of p53 expression by the continuously expressed endogenous E6 oncoprotein. Moreover, our study reveals that compromised Dkk1 expression is linked to increased resistance of HPV‐positive cervical cancer cells toward the proapoptotic activity of Cisplatin. Although Dkk1 can act as a Wnt antagonist, the antiapoptotic effect resulting from Dkk1 repression is not linked to an activation of this pathway. Rather, transcriptome and functional analyses uncover that Dkk1 repression leads to a strongly diminished stimulation of c‐Jun N‐terminal kinase (JNK) signaling which is required for efficient apoptosis induction by Cisplatin in cervical cancer cells. Further, we observed that Dkk1‐depleted cervical cancer cells induce senescence under Cisplatin treatment instead of apoptosis, suggesting that Dkk1 levels can strongly influence the phenotypic response of these cells toward Cisplatin. Collectively, these results provide new insights into the virus/host cell crosstalk in cervical cancer cells by identifying Dkk1 as a cellular target which is maintained under strong negative control by the continuous expression of the HPV oncogenes. Moreover, they identify Dkk1 as a critical determinant for the sensitivity of cervical cancer cells toward Cisplatin, showing that Dkk1 repression leads to increased Cisplatin resistance by impairing proapoptotic JNK signaling.

The impact of cycling hypoxia on the phenotype of HPV‐positive cervical cancer cells

AbstractCycling hypoxia (cycH) is a prevalent form of tumor hypoxia that is characterized by exposure of tumor cells to recurrent phases of hypoxia and reoxygenation. CycH has been associated with a particularly aggressive cellular phenotype of tumor cells and increased therapy resistance. By performing comparative analyses under normoxia, physoxia, chronic hypoxia, and cycH, we here uncover distinct effects of cycH on the phenotype of human papillomavirus (HPV)‐positive cervical cancer cells. We show that—other than under chronic hypoxia—viral E6/E7 oncogene expression is largely maintained under cycH as is the E6/E7‐dependent regulation of p53 and retinoblastoma protein. Further, cycH enables HPV‐positive cancer cells to evade prosenescent chemotherapy, similar to chronic hypoxia. Moreover, cells under cycH exhibit a particularly pronounced resistance to the proapoptotic effects of Cisplatin. Quantitative proteome analyses reveal that cycH induces a unique proteomic signature in cervical cancer cells, which includes a significant downregulation of luminal lysosomal proteins. These encompass the potentially proapoptotic cathepsins B and cathepsin L, which, however, appear not to affect the response to Cisplatin under any of the O2 conditions tested. Rather, we show that the proapoptotic Caspase 8/BH3‐interacting domain death agonist (BID) cascade plays a pivotal role for the efficiency of Cisplatin‐induced apoptosis in HPV‐positive cancer cells under all investigated O2 conditions. In addition, we provide evidence that BID activation by Cisplatin is impaired under cycH, which could contribute to the high resistance to the proapoptotic effects of Cisplatin. Collectively, this study provides the first insights into the profound phenotypic alterations induced by cycH in HPV‐positive cancer cells, with implications for their therapeutic susceptibility.

Revisiting the role of endogenous STAT3 in HPV‐positive cervical cancer cells

AbstractNovel treatment options for human papillomavirus (HPV)‐induced cancers are urgently required. The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is considered to be constitutively active in HPV‐positive cervical cancer cells and essential for their proliferation. Moreover, STAT3 was reported to undergo mutually stimulatory interactions with the HPV E6/E7 oncogenes. Thus, inhibiting STAT3 in HPV‐positive cancer cells is under discussion to provide a powerful novel therapeutic strategy. We here show that the antifungal drug ciclopirox destabilizes the STAT3 protein by acting as an iron chelator. However, by exploring the functional consequences of STAT3 inhibition in HPV‐positive cancer cells, we obtained several unexpected results. Chemical STAT3 inhibitors heterogeneously affect cervical cancer cell proliferation and those which act antiproliferative also block the growth of STAT3 knockout cells, indicating induction of off‐target effects. In contrast to several chemical inhibitors, genetic inhibition of STAT3 expression by either RNA interference or the CRISPR/Cas9 method does not appreciably affect cervical cancer cell proliferation. Transcriptome analyses indicate that blocking STAT3 expression in HPV‐positive cancer cells has very limited effects on putative STAT3 target genes. Although the targeted inhibition of specific growth‐promoting signaling pathways leads to a feedback activation of STAT3 in cervical cancer cells via Janus kinase 1/2, this does not lead to treatment resistance. Moreover, we did not obtain experimental evidence for a STAT3‐linked activation of HPV E6/E7 oncogene expression or, vice versa, an E6/E7‐dependent activation of STAT3, at endogenous conditions in cervical cancer cells. Collectively, these findings question the essential role of STAT3 in cervical cancer cell proliferation and the strategy to inhibit STAT3 in these cells for therapeutic purposes.