Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status

Abstract

Purpose: To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status.

Methods and materials: Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positive and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription-polymerase chain reaction for re-expression of reprogramming factors.

Results: Patients with HPV-positive tumors have superior overall survival and local-regional control. Human papillomavirus-positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus-negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors.

Conclusions: Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor.

Figure 1. Patients with HPV-positive HNSCC tumors have superior OS and local control

Kaplan-Meier estimates for OS (A) and locoregional control (B) for patients with HPV-positive (blue) or HPV-negative (red) HNSCC. 162 patients with locally advanced oropharyngeal HNSCC treated with primary chemoradiation or primary surgery followed by adjuvant radiation were followed to assess OS and time to local-regional recurrence. The patients were grouped based on p-16 positivity or -negativity.

Figure 2. Cells with low 26S proteasome activity in HNSCC lines have increased self-renewal

(A–B) Different cell lines of HNSCC contain a variable percentage of cells with low proteasome activity. Cells with low (ZsGreen-cODC-pos) and high (ZsGreen-cODC-neg) proteasome activity from three different HNSCC lines were sorted and the sphere forming capacity for these two populations was compared via primary (C) and secondary (D) sphere-forming assays. ZsGreen-cODC-pos cells had a significantly higher self-renewal capacity.

Figure 3. HPV-positive HNSCC lines have lower numbers of CSCs

(A) The frequency of ZsGreen-cODC-positive CSCs in the HPV-positive HNSCC lines is significantly lower compared to the HPV-negative lines (p<0.0001, unpaired t-test with Welch’s correction). (B) HPV-positive lines are significantly more radiosensitive compared to the HPV-negative lines at 2, 4, and 6Gy (statistical test for side panels: two-way ANOVA). (C) HPV-negative lines have an enhanced plating efficiency compared to the HPV-positive lines (p=0.0004, upaired t-test with Welch’s correction). (D) The frequency of intrinsic CSCs in HPV-positive and HPV-negative lines correlated well with their plating efficiency (top panel, r²=0.85), and surviving fraction at 2Gy (bottom panel, r²=0.68)

Figure 4. HPV-negative HNSCC cell lines lack cellular hierarchy due to enhanced spontaneous dedifferentiation

ZsGreen-cODC-positive and ZsGreen-cODC-negative cell populations from two different HPV-negative cell lines, Cal33 (A) and Fadu (B) were sorted and an in vivo limiting dilution assay was performed. ZsGreen-cODC-positive cells from Cal33 (A) were enriched ~3-fold in tumorigenic cells, while no enrichment was observed in the Fadu (B) line. The size of the tumors arising from the ZsGreen-cODC population Fadu cells were significantly larger at 5 weeks after innoculation (B, tumor sizes compared via two-way ANOVA). (C) ZsGreen-cODC-negative cells derived from HPV-negative lines spontaneously dedifferentiate into ZsGreen-cODC-positive cells at a significantly higher efficiency (p<0.0001, upaired t-test, with Welch’s correction) and the dedifferentiating rate correlates with the frequency of intrinsic stem cell numbers (D).

Figure 5. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation

ZsGreen-cODC-negative cells were sorted from four different HPV-negative HNSCC lines (A, left panel), and two HPV-positive lines (A, right panel), plated and irradiated. Radiation induced a significant (unpaired two-sided Student’s t-test) dose-dependent dedifferentiation of ZsGreen-cODC-negative cells into ZsGreen-cODC-positive cells in the HPV-negative lines (A, left panel). In contrast, the HPV-positive lines (A, right panel) dedifferentiated much less efficiently. (B) The self-renewal capacity of radiation-induced dedifferentiated cells after a single dose of 8Gy was compared to the non-irradiated cells in two different HPV-negative lines. (C and D) ZsGreen-cODC-neg cells were sorted, plated and irradiated with 0Gy or 8Gy. 5 days later the ZsGreen-cODC-neg and the induced ZsGreen-cODC-pos cells were re-sorted and qRT-PCR was performed for the Yamanaka reprogramming factors in both populations, in two HPV-neg lines, Fadu (C) and Cal33 (D).