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FBXO32, a member of the F-box protein family, is known to play both oncogenic and tumor-suppressive roles in different cancers. However, the functions and the molecular mechanisms regulated by FBXO32 in lung adenocarcinoma (LUAD) remain unclear. Here, we report that FBXO32 is overexpressed in LUAD compared with normal lung tissues, and high expression of FBXO32 correlates with poor prognosis in LUAD patients. Firstly, we observed with a series of functional experiments that FBXO32 alters the cell cycle and promotes the invasion and metastasis of LUAD cells. We further corroborate our findings using in vivo mouse models of metastasis and confirmed that FBXO32 positively regulates LUAD tumor metastasis. Using a proteomic-based approach combined with computational analyses, we found a positive correlation between FBXO32 and the PI3K/AKT/mTOR pathway, and identified PTEN as a FBXO32 interactor. More important, FBXO32 binds PTEN via its C-terminal substrate binding domain and we also validated PTEN as a bona fide FBXO32 substrate. Finally, we demonstrated that FBXO32 promotes EMT and regulates the cell cycle by targeting PTEN for proteasomal-dependent degradation. In summary, our study highlights the role of FBXO32 in promoting the PI3K/AKT/mTOR pathway via PTEN degradation, thereby fostering lung adenocarcinoma progression.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, has been implicated in preventing human pathologies, such as diabetes and cancer. However, the mechanisms underlying the beneficial effects of PLP are still unclear. Using Drosophila as a model system, we show that PLP deficiency, caused either by mutations in the pyridoxal kinase-coding gene (dPdxk) or by vitamin B6 antagonists, results in chromosome aberrations (CABs). The CAB frequency in PLP-depleted cells was strongly enhanced by sucrose, glucose or fructose treatments, and dPdxk mutant cells consistently displayed higher glucose contents than their wild type counterparts, an effect that is at least in part a consequence of an acquired insulin resistance. Together, our results indicate that a high intracellular level of glucose has a dramatic clastogenic effect if combined with PLP deficiency. This is likely due to an elevated level of Advanced Glycation End-products (AGE) formation. Treatment of dPdxk mutant cells with α-lipoic acid (ALA) lowered both AGE formation and CAB frequency, suggesting a possible AGE-CAB cause-effect relationship. The clastogenic effect of glucose in PLP-depleted cells is evolutionarily conserved. RNAi-mediated silencing of PDXK in human cells or treatments with PLP inhibitors resulted in chromosome breakage, which was potentiated by glucose and reduced by ALA. These results suggest that patients with concomitant hyperglycemia and vitamin B6 deficiency may suffer chromosome damage. This might impact cancer risk, as CABs are a well-known tumorigenic factor.

Overcoming resistance to therapy is a major challenge in castration-resistant prostate cancer (CRPC). Lineage plasticity towards a neuroendocrine phenotype enables CRPC to adapt and survive targeted therapies. However, the molecular mechanisms of epigenetic reprogramming during this process are still poorly understood. Here we show that the protein kinase PKCλ/ι-mediated phosphorylation of enhancer of zeste homolog 2 (EZH2) regulates its proteasomal degradation and maintains EZH2 as part of the canonical polycomb repressive complex (PRC2). Loss of PKCλ/ι promotes a switch during enzalutamide treatment to a non-canonical EZH2 cistrome that triggers the transcriptional activation of the translational machinery to induce a transforming growth factor β (TGFβ) resistance program. The increased reliance on protein synthesis creates a synthetic vulnerability in PKCλ/ι-deficient CRPC. The transition of androgen receptor-dependent prostate cancer to a therapy resistant cancer with neuroendocrine phenotype is an important process that remains poorly understood. Here, the authors show that PKCλ/ι-loss promotes epigenetic reprogramming resulting in a TGFβ resistance programme via transcriptional upregulation of translational machinery.

An intercentrosomal linker keeps a cell’s two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF βTrCP (Skp1–Cul1–F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by βTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication. Pagano and colleagues find that Plk1 and the E3 ubiquitin ligase SCF βTrCP mediate degradation of the centrosome cohesion protein Cep68 and show this mediates removal of Cep215 from the PCM and subsequent centriole separation in late mitosis.

Protein prenylation is believed to be catalyzed by three heterodimeric enzymes: FTase, GGTase1 and GGTase2. Here we report the identification of a previously unknown human prenyltransferase complex consisting of an orphan prenyltransferase α-subunit, PTAR1, and the catalytic β-subunit of GGTase2, RabGGTB. This enzyme, which we named GGTase3, geranylgeranylates FBXL2 to allow its localization at cell membranes, where this ubiquitin ligase mediates the polyubiquitylation of membrane-anchored proteins. In cells, FBXL2 is specifically recognized by GGTase3 despite having a typical carboxy-terminal CaaX prenylation motif that is predicted to be recognized by GGTase1. Our crystal structure analysis of the full-length GGTase3–FBXL2–SKP1 complex reveals an extensive multivalent interface specifically formed between the leucine-rich repeat domain of FBXL2 and PTAR1, which unmasks the structural basis of the substrate-enzyme specificity. By uncovering a missing prenyltransferase and its unique mode of substrate recognition, our findings call for a revision of the ‘prenylation code’.

The protein phosphatase 2A (PP2A) is a conserved heterotrimeric enzyme that regulates several cellular processes including the DNA damage response and mitosis. Consistent with these functions, PP2A is mutated in many types of cancer and acts as a tumor suppressor. In mammalian cells, PP2A inhibition results in DNA double strand breaks (DSBs) and chromosome aberrations (CABs). However, the mechanisms through which PP2A prevents DNA damage are still unclear. Here, we focus on the role of the Drosophila twins (tws) gene in the maintenance of chromosome integrity; tws encodes the B regulatory subunit (B/B55) of PP2A. Mutations in tws cause high frequencies of CABs (0.5 CABs/cell) in Drosophila larval brain cells and lead to an abnormal persistence of γ-H2Av repair foci. However, mutations that disrupt the PP4 phosphatase activity impair foci dissolution but do not cause CABs, suggesting that a delayed foci regression is not clastogenic. We also show that Tws is required for activation of the G2/M DNA damage checkpoint while PP4 is required for checkpoint recovery, a result that points to a conserved function of these phosphatases from flies to humans. Mutations in the ATM-coding gene tefu are strictly epistatic to tws mutations for the CAB phenotype, suggesting that failure to dephosphorylate an ATM substrate(s) impairs DNA DSBs repair. In addition, mutations in the Ku70 gene, which do not cause CABs, completely suppress CAB formation in tws Ku70 double mutants. These results suggest the hypothesis that an improperly phosphorylated Ku70 protein can lead to DNA damage and CABs.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, has been implicated in preventing human pathologies, such as diabetes and cancer. However, the mechanisms underlying the beneficial effects of PLP are still unclear. Using Drosophila as a model system, we show that PLP deficiency, caused either by mutations in the pyridoxal kinase-coding gene (dPdxk) or by vitamin B6 antagonists, results in chromosome aberrations (CABs). The CAB frequency in PLP-depleted cells was strongly enhanced by sucrose, glucose or fructose treatments, and dPdxk mutant cells consistently displayed higher glucose contents than their wild type counterparts, an effect that is at least in part a consequence of an acquired insulin resistance. Together, our results indicate that a high intracellular level of glucose has a dramatic clastogenic effect if combined with PLP deficiency. This is likely due to an elevated level of Advanced Glycation End-products (AGE) formation. Treatment of dPdxk mutant cells with α-lipoic acid (ALA) lowered both AGE formation and CAB frequency, suggesting a possible AGE-CAB cause-effect relationship. The clastogenic effect of glucose in PLP-depleted cells is evolutionarily conserved. RNAi-mediated silencing of PDXK in human cells or treatments with PLP inhibitors resulted in chromosome breakage, which was potentiated by glucose and reduced by ALA. These results suggest that patients with concomitant hyperglycemia and vitamin B6 deficiency may suffer chromosome damage. This might impact cancer risk, as CABs are a well-known tumorigenic factor.

The Mitotic Surveillance Pathway (MSP) monitors the duration of M-phase. Prolonged mitosis, caused by spindle attachment defects or microtubule-targeting drugs such as the taxane paclitaxel, induces the formation of the ternary \"mitotic stopwatch\" complex consisting of 53BP1, USP28, and p53. This event protects p53 from degradation, resulting in cell cycle arrest or apoptosis in daughter cells. In paclitaxel-resistant cancers, cells bypass the MSP, enabling unchecked proliferation and survival, although the underlying mechanisms remain unknown. Here, we demonstrate that 53BP1 physically interacts with GMCL1 but not its paralog, GMCL2, and we mapped the interaction regions on both proteins. CRL3 functions as a ubiquitin ligase that targets 53BP1 for degradation during M phase, impacting p53 levels in daughter cells. High GMCL1 expression significantly correlates with resistance to paclitaxel in cancer cell lines with wild-type p53, including endometrial, breast, and upper aerodigestive tract cancer cells. Loss of GMCL1 restores paclitaxel sensitivity in p53 expressing cells but not in p53 deficient cells. We propose that in cancers with high GMCL1 levels, the CRL3 -mediated degradation of 53BP1 prevents the formation of the mitotic stopwatch complex, leading to p53 degradation and sustained proliferation. Finally, our results indicate that GMCL1 inhibition represents a novel strategy to restore taxane sensitivity in resistant cancers.

Cells selectively remove damaged or excessive mitochondria through mitophagy, a specialized form of autophagy, to maintain mitochondrial quality and quantity. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation, and mitochondrial damage. However, the mechanisms by which cells remove specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4, an SKP1/CUL1/F-box protein ubiquitin ligase complex, localizes to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that, unlike wild-type FBXL4, pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13), do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism that actively suppresses mitophagy via preventing NIX and BNIP3 accumulation and propose that excessive basal mitophagy in the FBXL4-associated mtDNA depletion syndrome is caused by dysregulation of NIX and BNIP3 turnover. biorxiv;2022.10.12.511867v1/UFIG1F1ufig1