Metastases from primary breast cancer result in poor survival. βIII-tubulin (TUBB3) has been established as a therapeutic target for breast cancer metastases specifically to the brain. In this study, we conducted a systematic analysis to determine the regulation of TUBB3 expression in breast cancer metastases to the brain and strategically target these metastases using vinorelbine (VRB), a drug approved by the U.S. Food and Drug Administration (FDA). We found that human epidermal growth factor receptor 2 (HER2) signaling regulates TUBB3 expression in both trastuzumab-sensitive and trastuzumab-resistant neoplastic cells. We further discovered that bromodomain and extra-terminal domain (BET) inhibition increases TUBB3 expression, rendering neoplastic cells more susceptible to apoptosis by VRB. Orthotopic xenograft assays using two different breast cancer cell models revealed a reduction in tumor volume with BET inhibition and VRB treatment. In addition, in vivo studies using a model of multiple brain metastasis (BM) showed improved survival with the combination of radiation + BET inhibitor (iBET-762) + VRB (75% long-term survivors, P < 0.05). Using in silico analysis and BET inhibition, we found that the transcription factor myeloid zinc finger-1 (MZF-1) protein binds to the TUBB3 promoter. BET inhibition decreases MZF-1 expression and subsequently increases TUBB3 expression. Overexpression of MZF-1 decreases TUBB3 expression and reduces BM in vivo, whereas its knockdown increases TUBB3 expression in breast cancer cells. In summary, this study demonstrates a regulatory mechanism of TUBB3 and provides support for an application of BET inhibition to sensitize breast cancer metastases to VRB-mediated therapy.
BACKGROUND
While gliomas with hypermutated DNA are resistant to alkylating agents like temozolomide, they may be especially responsive to immunotherapy, since they express abundant neoantigens on their cell surfaces. Screening for hypermutated gliomas is currently being done through next-generation sequencing (NGS) panels that cover large portions of tumor DNA, although this is costly and access to such testing is not universal. Since hypermutated gliomas typically contain inactivating mutations in one of the main DNA mismatch repair (MMR) proteins, and cancers with an MMR mutation usually show loss of normal MMR protein, we sought to determine the feasibility of rapidly screening for hypermutated gliomas with an MMR immunohistochemistry (IHC) panel that is already in widespread use for colorectal adenocarcinomas.
METHODS
Tumor mutation burden (TMB) was determined via NGS for 101 gliomas, including 64 GBMs, 24 grade II-III astrocytomas, 9 grade II-III oligodendrogliomas, and 4 grade I gliomas. IHC for MSH2, MSH6, MLH1, and PMS2 was performed and analyzed on all gliomas while blinded to mutation profile and TMB.
RESULTS
Seven of 101 gliomas (7%) showed loss of an MMR protein by IHC. All 7 had matching MMR gene mutations and were hypermutated (100%), defined as TMB >20 per megabase of DNA. Of the remaining 94 with intact MMR IHC, only one was hypermutated. That case had an inactivating splice region mutation in another gene involved in DNA repair, ATM, although ATM is not part of the IHC panel originally developed for colorectal cancer. Overall sensitivity and specificity of the current MMR IHC panel for hypermutated gliomas was therefore 88% and 100%, respectively.
CONCLUSION
The colorectal MMR IHC panel, available in virtually all clinical IHC labs, is also a good screening test for hypermutated gliomas. Expansion of the panel to include even more DNA repair proteins, like ATM, would enhance its utility.
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