Pituitary tumors are the most common primary intracranial neoplasms. Humanin (HN) and Rattin (HNr), a rat homolog of HN, are short peptides with a cytoprotective action. In the present study, we aimed to evaluate whether endogenous HNr plays an antiapoptotic role in pituitary tumor cells. Thus, we used RNA interference based on short-hairpin RNA (shRNA) targeted to HNr (shHNr). A plasmid including the coding sequences for shHNr and dTomato fluorescent reporter gene was developed (pUC-shHNr). Transfection of somatolactotrope GH3 cells with pUC-shHNr increased apoptosis, suggesting that endogenous HNr plays a cytoprotective role in pituitary tumor cells. In order to evaluate the effect of blockade of endogenous HNr expression in vivo, we constructed a recombinant baculovirus (BV) encoding shHNr (BV-shHNr). In vitro, BV-shRNA was capable of transducing more than 80% of GH3 cells and decreased HNr mRNA. Also, BV-shHNr increased apoptosis in transduced GH3 cells. Intratumor injection of BV-shHNr to nude mice bearing s.c. GH3 tumors increased the number of apoptotic cells, delayed tumor growth and enhanced survival rate, suggesting that endogenous HNr may be involved in pituitary tumor progression. These preclinical data suggests that the silencing of HN expression could have a therapeutic impact on the treatment of pituitary tumors.

Humanin (HN) is a mitochondrial-derived peptide with cytoprotective effect in many tissues. Administration of Hn analogs has been proposed as therapeutic approach for degenerative diseases. Although HN has been shown to protect normal tissues from chemotherapy, its role in tumor pathogenesis is poorly understood. Here, we evaluated the effect of HN on the progression of experimental triple negative breast cancer (tnBc). the meta-analysis of transcriptomic data from the cancer Genome Atlas indicated that Hn and its receptors are expressed in breast cancer specimens. By immunohistochemistry we observed up-regulation of Hn in tnBc biopsies when compared to mammary gland sections from healthy donors. Addition of exogenous Hn protected tnBc cells from apoptotic stimuli whereas shRnA-mediated Hn silencing reduced their viability and enhanced their chemo-sensitivity. Systemic administration of HN in TNBC-bearing mice reduced tumor apoptotic rate, impaired the antitumor and anti-metastatic effect of chemotherapy and stimulated tumor progression, accelerating tumor growth and development of spontaneous lung metastases. These findings suggest that HN may exert pro-tumoral effects and thus, caution should be taken when using exogenous HN to treat degenerative diseases. In addition, our study suggests that HN blockade could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer. Breast cancer is the most common cause of death by cancer in women 1. Although new strategies have been developed for the treatment of breast tumors that express hormone receptors and/or human epidermal growth factor receptor 2 (Her2), there are no therapeutic options for patients with triple negative breast cancer (TNBC), for whom chemotherapy/radiotherapy remains the first-line treatment 2,3. Since these tumors frequently develop chemo-resistance 4 , novel therapeutic targets are urgently needed to improve the treatment of TNBC. A cytoprotective mitochondrial-derived peptide, humanin (HN), was discovered in healthy neurons of patients suffering Alzheimer's disease 5. HN was the first small open reading frame (ORF) identified within the mitochondrial DNA, encoded within the 16S rRNA gene (MT-RNR2) 6. HN can be translated both in the mitochondrial matrix or the cytosol, resulting in biologically functional 21 and 24-amino acid peptides, respectively 7. Small ORFs for six other small HN-like peptides (SHLPs) have been detected in the mitochondrial genome, two of which (SHLP2 and SHLP3) exhibit biological activity 8. Thirteen MT-RNR2-like loci that encode for fifteen HN-like peptides were detected in the nuclear genome 9. However, the expression of the mitochondrial gene MT-RNR2 is substantially higher than any nuclear isoform 9. HN regulates the mitochondrial apoptotic pathway by interaction with proteins of the Bcl-2 family 10. Intracellular HN binds to proapoptotic proteins, such as Bax, tBid and BimEL inhibiting the release of cytochrome