IntroductionAlthough tumor metastasis is the foremost cause of cancer-related death among breast cancer patients, druggable functional biomarker proteins and effective therapeutic strategies targeting the prevention of metastasis remain limited1. Furthermore, tumor cell metastasis is a multi-step process; at each step, the tumor cell must survive in a stressful environment, such as hypoxia, energy deprivation, or nutrient deprivation. Macroautophagy/autophagy is an evolutionarily conserved catabolic mechanism that is induced in response to various stresses and plays a critical role in each step of the metastatic cascade2,3,4,5. However, the mechanism by which autophagy regulates tumor metastasis remains unknown. Nevertheless, the autophagy process has been reported to have both pro- and anticancer properties, as uncontrolled autophagy induced by severe stress or many anticancer drugs can enhance autophagy and autophagic cell death3. Several recent publications have implicated autophagy associated with drug-resistant tumor cell survival, epithelial-mesenchymal transition (EMT) in tumor metastasis6, and inflammation7,8. Autophagy has a cytoprotective role in anoikis resistance9,10 and promotes the survival of dormant tumor cells11, resulting in metastatic growth. In addition, high expression of microtubule-associated protein 1A/1B-light chain 3B (LC3B), a central protein in autophagy in solid tumors, is associated with higher proliferative tumor cells, tumor metastasis, and poor outcome in cancer patients12, reinforcing that autophagy is a promising mechanism in tumor metastasis. However, induction of autophagy also results in the suppression of tumor progression and migration by various mechanisms, indicating that autophagy also has metastasis-preventing functions13,14. So far, targeting the autophagy mechanism associated with tumor cell survival is partially successful in the clinical setting15,16; thus, a molecular understanding of the regulatory mechanisms of autophagy in cancer is essential.Physiologically, autophagy is an essential homeostatic mechanism whereby dysfunctional protein aggregates or organelle components are engulfed in a double-membrane vesicle called an autophagosome and delivered to lysosomes for degradation and recycling to crucial cellular components17,18,19. Some tumor cells exhibit higher basal autophagy levels for their oncogenic events, even under normal conditions. Further, tumor cells depend more on autophagy than normal cells for an alternate energy source to cope with their metabolic demands20. The active unc-51 generally induces cellular canonical autophagy, such as the autophagy autophagy-activating kinase 1 (ULK1) protein complex; otherwise, autophagy signaling is blocked by the mechanistic target of rapamycin kinase (mTOR) complex 1 (mTORC1). Autophagy is mainly regulated by 5′ adenosine monophosphate-activated protein kinase (AMPK) downstream of the liver kinase LKB1, involving several cellular mechanisms in response to metabolic stress and maintaining energy homeostasis21. AMPK activation, together with mTORC1 inactivation in response to nutrient deprivation stress, directly phosphorylates the autophagy initiator protein kinase ULK1, thereby activating downstream components of the phosphoinositide 3-kinase class III Vps34 (also known as PIK3C3) complexes and recruiting the ATG14 complex, including Beclin-1, for LC3 lipidation (LC-II) and subsequent double-membrane structured autophagosome formation, a key event for the process of autophagy22,23. The AMPK signaling pathway coordinates cell-intrinsic autophagy and metabolism under low-energy conditions24.Nothing is known about the brain-specific serine/threonine protein kinases BRSK2 and BRSK1, the AMPK-related kinases, in regulating autophagy, particularly the mechanisms involved in autophagy-mediated tumor cell survival or autophagic cell death in response to nutrient-deprived stress. BRSK2 and BRSK1, also known as synapses of amphids defective (SAD) kinase SAD-A and SAD-B, respectively, downstream of the LKB1, are one of the dark/underinvestigated kinases recently defined by the National Institutes of Health (NIH) Illuminating the Druggable Genome (IDG) program; their functions are uncharacterized in cancer. BRSK2 and BRSK1 kinases are known to be activated by LKB1, PAK1, CAMKII, and PKA25,26,27. BRSK2 and BRSK1 kinases are mainly expressed in the human brain28 and result in neuronal polarization and brain development29. Furthermore, the loss of these kinases results in early neuronal apoptosis and a reduced number of progenitors, suggesting these kinases are required to survive cortical neurons30. Besides the brain, these kinases were also expressed at lower levels in the testis and pancreas25. BRSK2 promotes insulin secretion in response to glucose stimulation in pancreatic islets27,31,32.To date, similar to AMPK, BRSK2 kinase is induced by starvation to inhibit mTOR via phosphorylating the Tuberous Sclerosis Complex (TSC)-2, promote autophagy, and recent studies suggested that BRSK2 activation leads to activation of PI3K/AKT signaling31,33,34,35,36,37,38. Published results indicated that PI3K/AKT can be involved in both upstream and downstream regulation of mTORC139. Activation of mTORC1 strongly represses upstream PI3K/AKT signaling by the potent negative feedback loop and functions as a brake for AKT40. Therefore, new studies suggested that the repression of mTOR by starvation-induced BRSK2 may cause a loss of feedback inhibition on AKT activation34.However, the biological significance of BRSK2 kinase’s direct involvement in autophagy and cell fate in tumor cells in response to nutrient deprivation stress is unknown. Here, we identify the clinical relevance of BRSK2 expression in breast cancer patients. Furthermore, we have determined how specifically BRSK2 kinase directly regulates autophagy and is involved in tumor cell growth and survival in response to nutrient-deprived stress.ResultsElevated BRSK2 expression correlates with poor prognosis and disease recurrence in breast cancer patientsTo investigate the clinical relevance of the understudied serine/threonine kinase, BRSK2, in breast cancer, we have analyzed publicly available RNA-Seq datasets—i.e., The Cancer Genome Atlas (TCGA)41 and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC)42 cohorts—which include the gene expression profiles and clinical information of patients. BRSK2 protein has been reported as overexpressed in PDAC patients and is involved in PDAC cell survival34,43. First, our analyses depicted that the breast tissue expression levels of BRSK2 were significantly higher in the groups with cancer and relapsed or recurrent disease compared to the adjacent non-malignant normal tissue of patients (Fig. 1A, B). Importantly, BRSK2 expression in breast cancer patients appears to be correlated with the aggressive stage of breast cancer and marginally elevated in TNBC aggressive subtypes compared with ER + breast cancer patients Fig. 1C, D). We have also analyzed the survival rates of breast cancer patients (top vs. bottom thirds of patients as per normalized tumor gene expression) using the Kaplan–Meier method with a log-rank test. Disease-specific survival (DSS) and progression-free interval (PFI) or progression-free survival (PFS) in the TCGA breast cancer cohort (hazard ratio [HR] = 2.739, lower 95% confidence interval (CI) of HR = 1.643, upper 95% CI of HR = 4.566, p = 0.0002; HR = 1.489, lower 95% CI of HR = 1.005, upper 95% CI of HR = 2.205, p