Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair

Abstract

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 438C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of cH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to cH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear cH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.Trabectedin (Yondelis V R ) is a DNA-intercalating anticancer drug originally isolated from the sea squirt Ecteinascidia turbinata. The drug is established for the treatment of advanced soft-tissue sarcoma after failure of anthracyclines and ifosfamide as well as for the treatment of platinum-sensitive ovarial carcinoma in combination with liposomal doxorubicin. In sarcoma, trabectedin usually induces high rates of tumor growth arrest. In a worldwide-expanded access program investigating 1,895 patients, stable disease was reported in 43% and overall survival was 11.9 months. Unfortunately, treatment response with relevant tumor shrinkage was only seen in 5%. 1 Given the worse prognosis of advanced sarcoma, there is urgent need to improve treatment. Enhancing antitumor efficacy of trabectedin by a local treatment seems to be a promising strategy.

Heat-induced BRCA2 degradation in human tumours provides rationale for hyperthermia-PARP-inhibitor combination therapies

“…This provides a possible explanation for hyperthermia's sensitising effects towards irradiation and chemotherapeutic compounds that induce double strand breaks, such as cisplatin [34,35] and Trabectedin [36]. Moreover, attenuation of HR by hyperthermia sensitises cultured cells and mouse tumours to PARPinhibitors [19,[37][38][39], a class of therapeutics that specifically target cells deficient in HR.…”

Modulated Electro-Hyperthermia-Induced Tumor Damage Mechanisms Revealed in Cancer Models

“…Hyperthermic temperatures (41.8 °C or 43 °C) can also induce DNA damage, including double-strand breaks (DSB), as shown in human sarcoma (U2OS–osteosarcoma, SW872–liposarcoma, SW982-synovial sarcoma, RD-ES–Ewing sarcoma, and SKUT-1–leiomyosarcoma) and human colorectal carcinoma (DLD1) cell lines [ 68 ]. DSBs are indicated by the phosphorylation of histone 2AX (H2AX) at serine 139 observed, first after γ -irradiation, which is why it is marked as γ H2AX [ 47 ].…”

BRCA2 protects mammalian cells from heat shock

“…Heat shock affects stability/activity of several DNA repair factors: Ku70/80 [13,14], DNA-PK [10][11][12], MRE11/RAD50/ NBS1 [27][28][29], BRCA1 [30], BRCA2 [15][16][17][18][19][20] and other HR factors [31]. The NHEJ pathway was shown not to be involved in radio-sensitisation by heat [31][32][33].…”

“…The exact mechanisms by which hyperthermia inhibits NHEJ and HR have not been elucidated, but with NHEJ, it has been seen that heat shock lowers activity levels of DNA-PKcs [10][11][12] as well as protein levels of Ku70/80 [13,14], overall preventing proper functioning of the NHEJ pathway. In addition, BRCA2 is degraded by heat shock [15][16][17][18][19][20], likely leading to inhibition of HR; thus hyperthermia inhibits DNA repair for at least a certain period of time.…”