Expression of human papillomavirus oncoproteins E6 and E7 inhibits invadopodia activity but promotes cell migration in HPV‐positive head and neck squamous cell carcinoma cells

Kahue, Charissa N.; Jerrell, Rachel J.; Parekh, Aron

doi:10.1002/cnr2.1125

Cited by 3 publications

(4 citation statements)

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“…Nagel et al tested the migratory capacity of several OSCC cell lines using a wound-healing assay and showed that the migratory capacity of HPV-positive cell lines varies and is not signi cantly different from that of HPV-negative SCC cell lines [52]. Similarly, Kahue et al showed that the migratory capacity of HPV-positive OSCC cell lines does not signi cantly differ from the migratory capacity of HPV-negative OSCC cell lines; however, when the expression of E6 and E7 is abrogated in HPV-positive cell lines, their migratory capacity decreases, indicating a potential role of E6 and E7 in migration properties [53]. In our study, differences in cell migration were detected both in in vitro functional assays and at the transcriptome level.…”

Section: Discussionmentioning

confidence: 97%

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

Modic

Čemažar

Markelc

et al. 2023

Preprint

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Background Infection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, due to the species specificity of HPV, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo. Methods Two monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models. Results Characterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines. Conclusions In conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.

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Section: Discussionmentioning

confidence: 97%

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

Modic

Čemažar

Markelc

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Nagel et al tested the migratory capacity of several OSCC cell lines using a wound-healing assay and showed that the migratory capacity of HPV-positive cell lines varies and is not significantly different from that of HPV-negative SCC cell lines [ 60 ]. Similarly, Kahue et al showed that the migratory capacity of HPV-positive OSCC cell lines does not significantly differ from the migratory capacity of HPV-negative OSCC cell lines; however, when the expression of E6 and E7 is abrogated in HPV-positive cell lines, their migratory capacity decreases, indicating a potential role of E6 and E7 in migration properties [ 61 ]. In our study, differences in cell migration were detected both in in vitro functional assays and at the transcriptome level.…”

Section: Discussionmentioning

confidence: 99%

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

Modic¹,

Čemažar²,

Markelc³

et al. 2023

J Transl Med

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Background Infection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo. Methods Two monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models. Results Characterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines. Conclusions In conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.

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“…SCC-61 cells, an invasive HNSCC cell line originally acquired from the Yarbrough laboratory at our institution, have served as an established cell line model for invadopodia in our laboratory and were cultured as previously described (available from MilliporeSigma, Cat# SCC280, RRID: CVCL_7118). 20,21,24,25 Chemical inhibition of YAP was performed using verteporfin (Sigma) which inhibits YAP/TEAD interactions. 26,27 Kockdown (KD) of YAP was performed using a single ON-TARGETplus siRNA (sequences available from Dharmacon).…”

Section: Cell Culture and Reagentsmentioning

confidence: 99%

“…As previously described, statistics were performed using SPSS Software (IBM). 20,21,24,[33][34][35] All data were evaluated for normality with the Shapiro-Wilk or Kolmogorov-Smirnov test. Normal and non-parametric data were then analyzed by the Student's t-test or a Kruskal-Wallis test, respectively, with significance at a p-value < 0.05.…”

Section: Statisticsmentioning

confidence: 99%

A role for YAP-mediated regulation of invadopodia in HNSCC cells

Jerrell,

Marchlewski,

Parekh

2024

Preprint

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The objective of this study was to determine whether nuclear translocation of the transcriptional coactivator Yes-associated protein (YAP) was sensitive to extracellular matrix (ECM) rigidity and promoted Rho-associated kinase 2 (ROCK2) expression to affect invadopodia maturation and ECM degradation. ECM rigidity mimicking head and neck squamous cell carcinoma (HNSCC) tumor mechanical properties was simulated in vitro using a well-established model based on fibronectin-conjugated polyacrylamide gels (PAAs). The ratio of nuclear to cytoplasmic YAP and overall cellular ROCK2 levels were evaluated in HNSCC cells using quantitative immunofluorescence. YAP-mediated ROCK2 expression in HNSCC cells was determined using nested PCR and Western blot in response to the YAP inhibitor verteporfin. Invadopodia and ECM degradation were evaluated in HNSCC cells with siRNA-mediated inhibition of YAP using quantitative immunofluorescence in invadopodia assays. Both YAP nuclear translocation and ROCK2 cellular levels increased with ECM rigidity. Inhibition of YAP activity with verteporfin decreased ROCK2 gene and protein expression. Knockdown of YAP with siRNA inhibited the formation of mature invadopodia and ECM degradation but not total invadopodia (i.e., mature and immature or not degrading). Our study suggests that tumor-associated ECM rigidity can promote mechanically-induced transcriptional regulation to control proteolytic activity by affecting invadopodia maturation.

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Expression of human papillomavirus oncoproteins E6 and E7 inhibits invadopodia activity but promotes cell migration in HPV‐positive head and neck squamous cell carcinoma cells

Abstract: Background: The rapid increase in the incidence of head and neck squamous cell car-

Cited by 3 publications

References 146 publications

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

A role for YAP-mediated regulation of invadopodia in HNSCC cells

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