Piwirna-651 Expression Influences Treatment Response and Impacts Survival in Classical Hodgkin Lymphoma Patients through Regulation of ABCC5

Introduction: PiwiRNAs (piRNAs) are small non-coding RNAs processed by PIWI proteins, a subfamily of Argonaute proteins. The main functions of piRNAs include regulation of chromatin and genome organization, DNA methylation, transposon repression and regulation of protein synthesis. Until recently, PIWI/piRNA functions were believed to be limited to germinal stem cells and early embryonic development, where they play a crucial role in the regulation of genomic stability. However, piRNA expression has now been identified in some tumors, although the specific prognostic impact of piRNAs remains to be elucidated. We have examined the potential reactivation of the PIWI/piRNA pathway in classical Hodgkin lymphoma (cHL) and its impact on prognosis in cHL patients.

Methods: Formalin fixed and paraffin embedded tumor samples from 96 cHL patients were studied. All patients were diagnosed and treated in a single institution. HIV+ was an exclusion criterion. Median age was 33 years (range, 15–89); 47.9% were male; 30.2% were EBV+. The most frequent histological subtype was nodular sclerosis (65.6%). First-line treatment consisted of ABVD-type therapies in 90% of patients. 12 reactive lymph nodes (RLNs) were used as controls. The PIWI proteins PIWIL1 and PIWIL2 were studied by immunohistochemistry. The expression levels of three piRNAs (piR-651, piR-20365, piR-20582) were analyzed by Real-time PCR. cDNA was synthesized using miScript II RT Kit and Real-time PCR was performed using miScript SYBR Green PCR Kit (Qiagen). PiRNA expression was correlated with treatment response, disease-free survival (DFS) and overall survival (OS). Paired serum samples from seven prospectively collected cHL patients at diagnosis and at complete response and from 10 healthy controls were used to study the expression of piRNAs in serum. In addition, 5 cHL cell lines were used for the functional analysis. RNA hybrid, Renilla/Luciferase assay and Western Blot were used to identify and validate the piRNA targets. Statistical analyses were performed using R v2.13.

Results: PIWIL1 and PIWIL2 proteins were expressed in the cytoplasm of the Hodgkin/Reed Sternberg (HRS) cells, indicating that the piRNA pathway is active in cHL. This was corroborated by the finding that the 3 piRNAs were also expressed in all tumor samples and in the 5 cHL cell lines. piR-651 (p<0.0001) , piR-20365 (p=0.03) and piR-20582 (p<0.0001) were upregulated in cHL samples compared to the RLNs. Low expression levels of piR-651 were associated with lack of complete response to first-line treatment (p=0.006), shorter DFS (72.1 vs. 159.2 months, p=0.002) and shorter OS (87.6 vs. 154.5 months; p=0.010). The study of piR-651 expression in prospectively collected serum samples showed that piR-651 was underexpressed in cHL samples at diagnosis compared to serum from healthy controls (p=0.004). Interestingly, after complete remission piR-651 was overexpressed in serum (p=0.021) and the levels were similar to healthy controls.

In situ hybridization with LNA probes (Exiqon) showed that piR-651 was highly expressed in the cytoplasm of HRS cells. We studied putative targets genes of piR-651 using RNA hybrid. We identified several candidate genes related to drug metabolism, including ABCC5, an efflux pump of multiple drugs including doxorubicin. We cloned the 3’UTR region of ABCC5 in the psicheck2 vector and performed a Renilla/Luciferase assay. We observed a significant increase of 28% (p=0.047) in the Renilla luciferase levels in the cells transfected with 200nM of piR-651 inhibitor compared to oligo control. To further validate this, we transfected 3 HL cell lines with piR-651 inhibitor or oligo control and analyzed the protein levels of ABCC5 by Western Blot, observing a mean increase of 18% in the cells transfected with piR-651 inhibitor. These results showed that piR-651 inhibits the translation of ABCC5.

Conclusions:The PIWI/piRNA pathway is reactivated in cHL and the expression of piRNAs affects tumorogenesis and cHL patient outcome. piR-651 influences treatment response and survival through regulation of ABCC5. Our findings provide the groundwork for further investigation of the novel role of piRNAs in HL.

Acknowledgments: AECC-Catalunya (2014), APIF-UB (AC).