Mitotic Errors Increase Radiation Sensitivity in Multiple Cancer Cell Types and in Laryngeal Cancer Patients

Abstract

Chromosomal instability (CIN) arises from continual mitotic errors over the course of multiple cell divisions and is common in cancer. When CIN is increased beyond a maximally tolerated threshold it leads to cell death due to loss of both copies of one or more essential chromosomes. Combining two independent insults that each cause tolerable levels of CIN results in high CIN, cell death and tumor suppression. Because radiation causes CIN, we hypothesize that pre-existing CIN sensitizes tumor cells to radiation therapy. We characterized CIN in 4 HPV+ and 4 HPV- head and neck (HNC), 4 cervical, and 2 breast cancer cell lines before and 24 hours after 2 Gy of radiation. CIN was quantified by scoring lagging, bridge, and misaligned chromosomes, centrosome amplification, and multipolar spindles using immunofluorescence microscopy. To test our hypothesis, we created isogenic CIN and non-CIN HeLa and FaDu cells by knocking down the mitotic checkpoint protein Mad1, which induces lagging chromosomes, and by using MCF10A cells expressing tet-inducible polo-like kinase 4 (PLK4) to induce centrosome amplification. Clonogenic assays and murine tumor growth curves were used to determine radiation sensitivity in vitro and in vivo respectively. 6-chromosome FISH was used to quantify CIN and test if it was associated with local recurrence (LR) in a cohort of 29 laryngeal cancer patients treated with definitive (chemo)radiation. Significant differences were determined using a two-tailed student's t-test or Fisher's exact test. A total of 2 Gy of radiation significantly increased lagging, bridge, and misaligned chromosomes and induced centrosome amplification in all cancer cell types in vitro and in vivo. Most abnormal chromosomes after radiation were acentric fragments, which are known to missegregate. Mad1 knockdown in HeLa and FaDu cells increased CIN and led to significantly increased radiation sensitivity compared to their wild-type counterparts. PLK4 overexpression in MCF10A cells led to centrosome amplification and multipolar spindles, which also increased radiation sensitivity. Additionally, mitotic errors were significantly correlated with radiation response in both HPV+ and HPV- patient derived xenograft (PDX) HNC tumors. Finally, patients with laryngeal tumors with CIN below the median of the cohort had a 31% rate of LR after therapy, while tumors with CIN greater than or equal to the median had a LR rate of only 6%. Cancer cells with higher CIN are more sensitive to radiation therapy in vitro and in vivo. This was true for different types of mitotic errors and in different cancer models implying this is likely tumor type agnostic. Importantly, laryngeal tumors with higher baseline levels of CIN had an improved response to definitive radiation therapy, implying increased radiation sensitivity. These studies have potential implications for personalization of radiation dose.