Blocking autophagy enhanced cytotoxicity induced by recombinant human arginase in triple-negative breast cancer cells

Depletion of arginine by recombinant human arginase (rhArg) has proven to be an effective cancer therapeutic approach for a variety of malignant tumors. Triple-negative breast cancers (TNBCs) lack of specific therapeutic targets, resulting in poor prognosis and limited therapeutic efficacy. To explore new therapeutic approaches for TNBC we studied the cytotoxicity of rhArg in five TNBC cells. We found that rhArg could inhibit cell growth in these five TNBC cells. Intriguingly, accumulation of autophagosomes and autophagic flux was observed in rhArg-treated MDA-MB-231 cells. Inhibition of autophagy by chloroquine (CQ), 3-methyladenine (3-MA) and siRNA targeting Beclin1 significantly enhanced rhArg-induced cytotoxic effect, indicating the cytoprotective role of autophagy in rhArg-induced cell death. In addition, N-acetyl-l-cysteine (NAC), a common antioxidant, blocked autophagy induced by rhArg, suggesting that reactive oxygen species (ROS) had an essential role in the cytotoxicity of rhArg. This study provides new insights into the molecular mechanism of autophagy involved in rhArg-induced cytotoxicity in TNBC cells. Meanwhile, our results revealed that rhArg, either alone or in combination with autophagic inhibitors, might be a potential novel therapy for the treatment of TNBC.Breast cancer, the most common cause of cancer death in women, is a kind of complex and heterogeneous neoplasm.¹ Approximately 15% of breast carcinomas are triple-negative breast cancers (TNBCs), which have high rates of recurrences and mortality.² TNBCs are defined by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor type 2 (HER2). These tumors are characterized by clinically aggressive behaviors, high recurrence rate and poor prognosis. Owing to lack of targeted therapies (such as hormone therapy or anti-HER2 therapy), currently chemotherapy is the primary treatment for TNBC.³ Therefore, investigating new therapeutic approaches is urgently needed for improving the clinical outcome of TNBC therapy.Recently, deprivation of l-arginine has been a potential therapeutic method for cancers.⁴ By culturing cells in the arginine-free media, a variety of human cancer cells have been found to be auxotrophic for arginine, depletion of which resulted in cell death. Importantly, recombinant human arginase (rhArg) has shown potent anticancer effect in acute myeloid leukemia and acute lymphoblastic T-cell leukemia and solid tumors in vitro and in vivo^{5, 6, 7, 8, 9} and is currently under clinical investigation for the treatment of melanoma¹⁰ and hepatocellular carcinoma (HCC).¹¹ These carcinomas are auxotrophic for arginine, mainly because of the absence of arginine endogenous synthetical pathway. However, there are no reports about the efficiency in the therapy of breast cancer by rhArg through depletion of arginine.An increasing number of studies have shown that autophagy is stimulated in response to external stressors (such as starvation and oxidative stress) and internal needs (for example, removal of aggregate-prone proteins).¹² Autophagy is an evolutionarily conserved catabolic process responsible for the routine degradation of bulk superfluous or dysfunctional proteins and organelles.¹³ Autophagy serves as a protective role in response to a majority of anticancer drugs and in the pathogenesis process.^{14, 15} Not surprisingly, the relationship between autophagy and apoptosis, both genetically regulated and evolutionarily conserved, is complex, and appears to be related to cellular contexts.¹⁶ Meanwhile, mounting evidence accumulated has revealed that autophagy stimulation and reactive oxygen species (ROS) are closely linked in response to cancer therapeutics.^{17, 18} Notably, the essential contribution of mitochondrially generated ROS in the modulation of autophagy during starvation has been highlighted.In this study, we investigated whether rhArg might be a potential therapy for TNBC. We reported for the first time that rhArg-induced cell growth inhibition and caspase 3-independent apoptosis in MDA-MB-231 cells. Also, we found that rhArg could induce autophagy in MDA-MB-231 cells in a dose- and time-dependent manner. Interestingly, blocking autophagy potentiated cytotoxicity induced by rhArg, indicating that autophagy had a cytoprotective role in the treatment of rhArg. Meanwhile, ROS was involved in the autophagy and cell growth inhibition induced by rhArg. With our findings mentioned above, rhArg has shown potential to be a promising therapy for TNBC. Furthermore, the combination with autophagy-targeting drugs displayed multipronged treatment for breast cancer therapy.