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

Cycling 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.

Novel 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.

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

Oncogenic 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.

FAM57A (Family with Sequence Similarity 57 Member A) Is a Cell-Density-Regulated Protein and Promotes the Proliferation and Migration of Cervical Cancer Cells.

The FAM57A (family with sequence similarity 57 member A) gene is controversially discussed to possess pro- or anti-tumorigenic potential. Here, we analyze the regulation of cellular FAM57A protein levels and study the functional role of FAM57A in HPV-positive cervical cancer cells. We find that FAM57A protein expression strongly depends on cell density, with FAM57A being readily detectable at low cell density, but undetectable at high cell density. This regulation occurs post-transcriptionally and is not mirrored by corresponding changes at the RNA level. We further show that FAM57A protein levels are highly increased in cervical cancer cells cultivated at hypoxia compared to normoxia and provide evidence that FAM57A is a hypoxia-responsive gene under control of the α-subunit of the HIF-1 (hypoxia-inducible factor-1) transcription factor. Yet, the strong relative increase of FAM57A protein levels in hypoxic cells is predominantly cell-density-dependent and occurs post-transcriptionally. Other anti-proliferative effectors besides hypoxia, such as silencing of HPV E6/E7 oncogene expression in cervical cancer cells, also result in an increase of FAM57A levels compared to untreated cells. Functional analyses reveal that FAM57A repression leads to pronounced anti-proliferative as well as anti-migratory effects in cervical cancer cells. Taken together, these results provide insights into the regulation of FAM57A protein levels and reveal that they underlie a tight cell-density-dependent control. Moreover, they identify FAM57A as a critical determinant for the phenotype of cervical cancer cells, which promotes their proliferation and migration capacities.

CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4.

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.