Modulated electro‐hyperthermia suppresses H19, a tumor promoting long non coding RNA, in a triple‐negative breast cancer model

Introduction Modulated electro‐hyperthermia (mEHT) is a complementary antitumor therapy, based on selective tumor cell killing by a 13.56 MHz radiofrequency induced electric field. The H19 long non coding RNA (lncRNA) is involved in tumor progression and metastasis. It’s overexpression is associated with poor prognosis and therapy resistance in breast cancer. We observed previously significant tumor inhibitory effects of mEHT. Aims Our aim was to investigate the hypothesis, that mEHT effects are related to H19 lncRNA inhibition in triple negative breast cancer (TNBC) spheroids and in a TNBC bearing mouse model. Methods 4T1 spheroids were embedded in Matrigel® and treated with a single mEHT, conventional hyperthermia (cHT) or normothermia (Ctr) for 30 minutes. Spheroids were collected 24 hours after treatment, RNA was isolated and processed for Real‐Time PCR (RT‐PCR). TNBC cells (highly aggressive 4T1 or less aggressive 4T07) were inoculated orthotopically in female BALB/c mice. Tumor growth was monitored in vivo by digital caliper and ultrasound (Phillips Sonos 5500), mice were randomized into two groups based on tumor size. Mice were treated with mEHT in monotherapy or in combination with methotrexate (MTX) 2 or 3 times for 30 minutes with 0.7±0.3W power to achieve 40°C skin temperature above the tumor. At the end of the experiments mice were euthanized, the tumors were dissected and processed for molecular biologic techniques. H19 expression was measured with RT‐PCR, results were normalized to GAPDH. Results Single mEHT or cHT treatment of 4T1 spheroids reduced significantly H19 expression compared to a normothermic control (Ctr:0.004±0.0004, cHT:0.001±0.0001 mEHT:0.0006±0.0002, p<0.0001). mEHT decreased H19 expression more effectively, than conventional HT (cHT:0.001±0.0001 mEHT:0.0006±0.0002, p<0.01). There was a significant decrease in H19 expression of 4T1 tumors in vivo after two (sham:0.068±0.044, mEHT:0.033±0.024, p<0.05) and three mEHT treatments (sham:0.097±0.059 vs mEHT:0.050±0.030, p<0.05) compared to the sham group. In case of combination treatments H19 expression was significantly lower in the mEHT+MTX group compared to MTX only (MTX:0.104±0,038 vs mEHT+MTX:0.056±0.025, p<0.01). The basic expression of H19 was significantly lower in 4T07 tumors compared to the more aggressive 4T1 tumors (4T07:0.006±0.004 vs 4T1:0.399±0.071, p<0.0001). In 4T07 tumors H19 expression did not change after three mEHT treatments (sham:0.404±0.334 vs mEHT:1.391±1.840, p>0.10) compared to the sham group. Conclusion Our results demonstrate, that modulated electro‐hyperthermia can reduce the expression of tumor promoting H19 lncRNA in vitro and in vivo both in monotherapy and in combination with chemotherapy. Our findings suggest, that mEHT as an alternative complementary treatment could promote antitumor therapy by inhibiting the tumor progression mediating H19 lncRNA expression. More effective therapy against the more aggressive 4T1 line may be related to higher baseline expression and more significant effect on H19. Support or Funding Information Grant NVKP_16‐1‐2016‐0042, EFOP‐3.6.3‐VEKOP‐16‐2017‐00009