CDKN1B/p27 regulates autophagy via the control of Ragulator and MTOR activity in amino acid-deprived cells

The tumor suppressor CDKN1B/p27 Kip1 binds to and inhibits cyclin-CDK complexes in the nucleus, inducing cell cycle arrest. However, when in the cytoplasm, CDKN1B may promote tumorigenesis. Notably, cytoplasmic CDKN1B was reported to promote macroautophagy/autophagy in response to nutrient shortage by a previously unknown mechanism. In our recent work, we found that during prolonged amino acid starvation, CDKN1B promotes autophagy via an MTORC1-dependent pathway. A fraction of CDKN1B translocates to lysosomes, where it interacts with the Ragulator subunit LAMTOR1, preventing Ragulator assembly, which is required for MTORC1 activation in response to amino acids. Therefore, CDKN1B represses MTORC1 activity, leading to nuclear translocation of the transcription factor TFEB and activation of lysosomal function, enhancing starvation-induced autophagy flux and apoptosis. In contrast, cells lacking CDKN1B survive starvation despite reduced autophagy, due to elevated MTORC1 activation. These findings reveal that, by directly repressing MTORC1 activity, CDKN1B couples the cell cycle and cell growth machineries during metabolic stress. acid starvation. Previous studies suggested the presence of CDKN1B on lysosomal and autophagy compartments via interactions with either SNX6 (sorting nexin 6) or SQSTM1/p62, but not in the context of metabolic stress. Importantly, CDKN1B was shown previously to bind to p18/p27RF-Rho, which was later renamed LAMTOR1, a lysosomal scaffold part of the Ragulator complex acting as an anchor and a GAP for RRAG GTPases that recruit MTORC1 to lysosomal membrane prior to its activation in response to amino acids. Our results indicate that amino acid starvation promotes binding of CDKN1B to LAMTOR1 on lysosomes, thereby preventing the assembly of Ragulator and the translocation of RRAGs and MTORC1 to lysosomes, therefore maintaining MTORC1 in an inactive state. Consequently, TFEB, a master regulator of lysosome and autophagy gene expression that is kept inactive by MTORC1, enters the nucleus to drive expression of genes required for activation of lysosomal functions and degradation of autophagic cargo. In contrast, increased MTORC1 activity in cdkn1b -/- cells sequesters TFEB in the cytoplasm,

These findings reveal that, by directly repressing MTORC1 activity, CDKN1B couples the cell cycle and cell growth machineries during metabolic stress.

Main text
Every proliferating cell must ensure that it has enough mass and energy prior to division.
Therefore, cells must rapidly respond to continuously changing nutrient levels, activating kinase cascades that promote either anabolic reactions, leading to cell growth, or catabolic reactions, such as autophagy, which are associated with energy production. The MTORC1 pathway adapts cell metabolism to environmental conditions by promoting cell growth and proliferation and inhibiting autophagy when nutrients are plentiful. Whereas MTORC1 regulation has been extensively studied in the context of acute increase of nutrient levels, little is known about how cells coordinate their metabolism in conditions of prolonged starvation, to which cancer cells are often exposed because of poor tumor vascularization.
Previous studies showed that the cell cycle inhibitor CDKN1B/p27 Kip1 contributes to the response of cancer cell to metabolic stress and that CDKN1B status determines whether cells survive prolonged serum and/or glucose starvation or undergo apoptosis.
CDKN1B is considered as a tumor suppressor due to its anti-proliferative activity via inhibition of cyclin-CDK complexes. However, in some cancers, CDKN1B may promote tumorigenesis via CDK-independent mechanisms, as shown in CDKN1B knockin mice in which CDKN1B cannot bind to and inhibit cyclin-CDKs (CDKN1B/p27 CK-). CDKN1B shuttles between the nucleus and cytoplasm, and cytoplasmic localization of CDKN1B is associated with poor prognosis in many types of cancers, suggesting that CDKN1B exhibits its pro-oncogenic functions outside the nucleus. Importantly, previous studies showed that in response to starvation, CDKN1B relocalizes to the cytoplasm and promotes autophagy, but neither the subcellular compartment in which CDKN1B localizes in starved cells, nor the molecular mechanism underlying the pro-autophagic role of CDKN1B was investigated in detail.
Using mouse embryonic fibroblasts (MEFs) isolated from Cdkn1b +/+ and cdkn1b -/mice, we confirmed the pro-autophagic role of CDKN1B in cells deprived of amino acids for prolonged periods of time (>24 h) [1]. Surprisingly, Cdkn1b +/+ cells are more susceptible to apoptosis induced by amino acid deprivation than cdkn1b -/cells, despite reduced autophagy in the latter.  Surprisingly, during prolonged amino acid starvation, increased autophagy flux in Cdkn1b +/+ cells does not protect against apoptosis and, in fact, we found that general inhibition of ATG5-dependent autophagy does not affect apoptosis rate in these conditions.
While the identification of a CDKN1B-MTORC1-TFEB axis provides an explanation for the autophagy defect in cells lacking CDKN1B, it raises questions about the ability of cdkn1b -/to survive long-term starvation. As lysosomal function is significantly reduced, it is unlikely that autophagy or macropinocytosis, which both require an intact lysosomal machinery, support their metabolism. Therefore, an attractive hypothesis under investigation is that the absence of CDKN1B triggers a metabolic reprogramming, similar to that described in deep quiescent cells, which are also characterized by enhanced MTORC1 signaling, impaired autophagy and accumulation of non-functional lysosomes. This metabolic reprogramming consists of decoupling metabolic pathways from nutrient availability, enabling cells to carry out anabolic reactions even when nutrients are limited. Therefore, the recruitment of CDKN1B to lysosomal membranes may be seen as an adaptive mechanism protecting cells from uncontrolled growth and proliferation under adverse conditions. Thus, in contrast to previously described oncogenic roles of cytoplasmic CDKN1B, lysosomal CDKN1B may act as a tumor suppressor, and support the idea that the role of CDKN1B is determined by its binding partners and subcellular localization, themselves dictated by environmental conditions.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.