Pituitary tumors (PTs): PTs are common, occurring in almost 17% of the population over the lifetime (Daly et al., 2006; Fernandez et al., 2010), and they represent approximately 10% of all intracranial tumors (Ezzat et al., 2004). They can cause significant morbidity because of hormonal abnormalities and symptoms such as headaches and visual disturbances. Standard treatment options, including surgery, medical therapy, and radiotherapy, often fail to control the hormone excess and/or inhibit tumor growth, in particular the aggressive PTs. Whereas treatment with the chemotherapeutic drug, temozolomide (TMZ), in aggressive ACTH-producing PTs, initially may lead to short-term clinical benefits, long-term therapeutic efficacy occurs only in a limited number of cases (Takeshita et al., 2009; Bush et al., 2010; Rotondo et al., 2012; Mohammed et al., 2009; Raverot et al., 2010; Moshkin et al., 2011; Annamalai et al., 2012; Jouanneau et al., 2012; Bengtsson et al., 2015; McCormack et al., 2018; Burman et al., 2022). The addition of capecitabine, a prodrug of 5-fluorouracil (5-FU), to TMZ (CAPTEM) has been effective in a selected group of aggressive PTs (Nakano-Tateno et al., 2020, 2021). Whether CAPTEM is superior to treatment with TMZ is not known. Nonetheless, this combination therapy was supported by our experiments using mouse ACTH-producing PT cells, AtT-20 cells (Nakano-Tateno et al., 2020). As many aggressive PTs are resistant to CAPTEM, establishing new therapeutic options is required.

Autophagy inhibitors and PTs: Autophagy is essential for survival in eukaryotic cells. The products from autophagic processes, such as amino acids, are recycled to maintain cellular functions (Galluzzi et al., 2015). Autophagy can enhance tumor progression and lead to resistance to medical therapy. Reduced intracellular amino acids concentration induces mTOR1 dephosphorylation, leading to activation of microphthalmia/transcription factor E (MiT/TFE)-dependent gene expression followed by autophagosome formation (La Spina et al., 2021; Raben and Puertollano, 2016; Perera et al., 2019). Among these transcription factor family genes, TFE3 has pivotal roles in cancer metastasis as well as enhanced longevity by caloric restriction (Kepp et al., 2020). Therefore, inhibition of autophagy may provide a novel therapeutic approach to cancer (Jogalekar et al., 2021; Xu et al., 2018). Bafilomycin A1 (BFA) is a macrolide antibiotic known to inhibit autophagy flux by preventing vacuolar-type H + -ATPase (v-ATPase)-driven acidification of endosomes and lysosomes. Inhibition of autophagy by BFA enhances the effects of chemotherapy in cancer cells (Liu et al., 2015; Li et al., 2016; Xie et al., 2014; Greene et al., 2013). Chroloquine (CQ), an FDA-approved anti-malarial drug, has been shown to suppress the binding of autophagosomes to lysosomes and enhance anti-tumor effects of chemotherapeutic agents and radiation therapy (Sotelo et al., 2006; Sasaki et al., 2010; Selvakumaran et al., 2013; Cerniglia et al., 2012; Bhat et al., 2018). A recent meta-analysis demonstrated that chemotherapy or radiotherapy in combination with CQ is superior to monotherapy in cancer treatment (Xu et al., 2018). Further, of particular relevance to the present study, was a previous report that showed an additive effect of CQ and cabergoline on two PT cell lines, the growth hormone (GH)-producing GH3 cells, and the prolactin-producing PRL235 cells (Lin et al., 2017). However, the effects of CQ and other autophagy inhibitors on ACTH production and cell growth in the mouse ACTH-producing PT cells, AtT-20 cells, have not been assessed. Moreover, in support of the importance of autophagy on PT cell growth and hormone production, several PT-targeting drugs, such as cabergoline, bromocriptine, and octreotide, probably reduced cell growth through inhibition of autophagy (Lin et al., 2017; Geng et al., 2017; Tulipano and Giustina, 2020).

Therefore, in this study, we tested the role of autophagy inhibitors in suppressing cell growth and hormone production using two in vitro models of PTs. We also investigated whether the combination of CQ and TMZ had additive tumor suppressive effects in PT cells.