The FBXW7‐NOTCH interactome: A ubiquitin proteasomal system‐induced crosstalk modulating oncogenic transformation in human tissues

Abstract Background Ubiquitin ligases or E3 ligases are well programmed to regulate molecular interactions that operate at a post‐translational level. Skp, Cullin, F‐box containing complex (or SCF complex) is a multidomain E3 ligase known to mediate the degradation of a wide range of proteins through the proteasomal pathway. The three‐dimensional domain architecture of SCF family proteins suggests that it operates through a novel and adaptable “super‐enzymatic” process that might respond to targeted therapeutic modalities in cancer. Recent findings Several F‐box containing proteins have been characterized either as tumor suppressors (FBXW8, FBXL3, FBXW8, FBXL3, FBXO1, FBXO4, and FBXO18) or as oncogenes (FBXO5, FBXO9, and SKP2). Besides, F‐box members like βTrcP1 and βTrcP2, the ones with context‐dependent functionality, have also been studied and reported. FBXW7 is a well‐studied F‐box protein and is a tumor suppressor. FBXW7 regulates the activity of a range of substrates, such as c‐Myc, cyclin E, mTOR, c‐Jun, NOTCH, myeloid cell leukemia sequence‐1 (MCL1), AURKA, NOTCH through the well‐known ubiquitin‐proteasome system (UPS)‐mediated degradation pathway. NOTCH signaling is a primitive pathway that plays a crucial role in maintaining normal tissue homeostasis. FBXW7 regulates NOTCH protein activity by controlling its half‐life, thereby maintaining optimum protein levels in tissue. However, aberrations in the FBXW7 or NOTCH expression levels can lead to poor prognosis and detrimental outcomes in patients. Therefore, the FBXW7‐NOTCH axis has been a subject of intense study and research over the years, especially around the interactome's role in driving cancer development and progression. Several studies have reported the effect of FBXW7 and NOTCH mutations on normal tissue behavior. The current review attempts to critically analyze these mutations prognostic value in a wide range of tumors. Furthermore, the review summarizes the recent findings pertaining to the FBXW7 and NOTCH interactome and its involvement in phosphorylation‐related events, cell cycle, proliferation, apoptosis, and metastasis. Conclusion The review concludes by positioning FBXW7 as an effective diagnostic marker in tumors and by listing out recent advancements made in cancer therapeutics in identifying protocols targeting the FBXW7‐NOTCH aberrations in tumors.

Recent findings: Several F-box containing proteins have been characterized either as tumor suppressors (FBXW8, FBXL3, FBXW8, FBXL3, FBXO1, FBXO4, and FBXO18) or as oncogenes (FBXO5, FBXO9, and SKP2). Besides, F-box members like βTrcP1 and βTrcP2, the ones with context-dependent functionality, have also been studied and reported. FBXW7 is a well-studied F-box protein and is a tumor suppressor.
NOTCH signaling is a primitive pathway that plays a crucial role in maintaining normal tissue homeostasis. FBXW7 regulates NOTCH protein activity by controlling its halflife, thereby maintaining optimum protein levels in tissue. However, aberrations in the FBXW7 or NOTCH expression levels can lead to poor prognosis and detrimental outcomes in patients. Therefore, the FBXW7-NOTCH axis has been a subject of intense study and research over the years, especially around the interactome's role in driving cancer development and progression. Several studies have reported the effect of FBXW7 and NOTCH mutations on normal tissue behavior. The current review attempts to critically analyze these mutations prognostic value in a wide range of tumors. Furthermore, the review summarizes the recent findings pertaining to the FBXW7 and NOTCH interactome and its involvement in phosphorylation-related events, cell cycle, proliferation, apoptosis, and metastasis. ligase is the largest family of ubiquitin ligases, which facilitate the turnover of approximately 20% of all ubiquitin-proteasomal system (UPS)-regulated proteins in the mammalian system, including those critically involved in tumorigenesis. The SCF family of ubiquitin ligases (SCFCdc4) was first studied in budding yeast (Saccharomyces cerevisiae) by using the in vitro reconstitution technique. Thereafter, its complete structure was deciphered, and SCF was found to be a variable complex made up of S-phase kinase-associated protein 1 (Skp-1), Cullins, F-box proteins, and RBX/ROC RING finger proteins ( Figure 1). Cullins act as a scaffold and comprises sequences that bind Skp-1 and F-box proteins at its N-terminus and the RING proteins at its C-terminus. The RING protein family consists of only two evolutionary conserved members, RBX1 (RING box protein 1), also known as ROC1 (regulator of Cullins), and RBX2/ROC2 (also known as SAG [sensitive to apoptosis gene]) that are necessary for driving the catalytic activity of SCF. F-box proteins are the most critical element of the SCF assembly that is tasked with defining the substrate specificity of the SCF complex. The mammalian genome comprises 69 F-box proteins that include the WD40 domain-containing FBXWs, leucinerich repeats-containing FBXLs and FBXOs (made up of diverse domains), and 7 Cullins (Cul-1, 2, 3, 4A, 4B, 5, and 7). FBXW7 is the most widely studied F-box protein due to its role in a panel of both normal and malignant cellular processes. FBXW7 binds to Skp1 and Cullins through its F-box domain, and to the substrates through WD40 or leucine-rich domains. Overall, Cullins-RBX/ROC acts as the core of the SCF complex where RBX binds to ubiquitin-conjugating enzymes (E2) and moderate the ubiquitin transfer from E2 to F-boxtargeted substrates. The activity of SCF E3 ligases is also driven by events such as Cullin neddylation that disturbs the inhibitory binding of Cullin by Cullin-associated NEDD8-dissociated protein 1 (CAND1).
Altogether, Cullin-based SCF assembly can be divided into four categories, Cul1-Skp1-F-box, Cul2/5-Elongins-B/C-VHL/SOCS box, Cul3-BTB, and Cul4A/B-DDB1-DWD, thereby making SCF the largest family of E3 ligases. [1][2][3] FBXW7 (F-box with seven tandem WD40; also known as Fbw7, Sel-10, hCdc4, hAgo, or Archipelago), encoded by the gene FBXW7 (loci, 4q31q.3), was first isolated as Cdc4 in budding yeast. The gene comprises 4 untranslated regions (UTRs) and 13 coding exons, which altogether define the substrate specificity of FBXW7, primarily through alternate splicing mechanisms. The human FBXW7 gene is reportedly deleted in approximately 30% of all cancer types, thereby underscoring its role in tumor progression and prognosis. Mammalian cells express three FBXW7 isoforms, FBXW7α, FBXW7β, and FBXW7γ, and each of these isoforms is localized in a tissue-specific manner to restrict their interaction to specific substrates. Broadly, FBXW7α is found in abundance in the nucleoplasm, as compared to FBXW7β, which is cytoplasmic and FBXW7γ that is nucleolar.  4,5 Activated GSK3 phosphorylates the CPDs present in target substrates, which subsequently bind to the WD40 repeats of FBXW7. GSK3-mediated phosphorylation of CPD is an essential step in the interaction between the substrate and FBXW7, although the role of other kinases in the process is now being explored. Mutations within the CPD motif have been observed in several cancers that lead to the stabilization of oncogenic substrates, like c-Myc and NOTCH. Sequence analysis of FBXW7 substrates has identified the conserved CDP motif to be ΦXΦΦΦ-T/S-PPX-S/T/E, where Φ is a hydrophobic residue and X could be any amino acid. It must be noted that the phosphorylation of S/T/E residue triggers the GSK3-mediated phosphorylation of T/S residue.
Some reports suggest the existence of more than one CPD motif within FBXW7 substrates, like Cyclin E that comprise two CPD motifs, one located in the T380 position, and the other being centered around T62. Together, these two CPD motifs improve the efficiency of FBXW7 binding with Cyclin E in response to various signaling cues. 6 Some studies highlight the antagonistic behavior of FBXW7α and FBXW7β toward substrates. For instance, peroxisome proliferator-activated receptor-gamma co-activator (PGC)-1α, a transcriptional co-activator with widespread effects on cellular energy metabolism undergoes different fate under FBXW7α and FBXW7β, respectively. Cellular PGC-1α is downregulated by FBXW7β while F I G U R E 1 Domain architecture of E3 ubiquitin ligase and SCF-type E3 ligase complex: A, common architecture underlies E3 ubiquitin ligase complex that mediate the targeted degradation of many cellular proteins. In targeting substrate proteins for degradation, ubiquitin is passed from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme to the protein substrate, with the final step (ligating ubiquitin to the substrate) catalyzed by an E3 ubiquitin ligase. B, The SCF complexes are known to be E3 ligases and each complex in SCF-type E3 ligase interacts with a set of adapter proteins that recruit different binding partners through specific protein-protein interaction domains such as WD40 repeats and Leucine-rich repeats (LRR) and facilitate substrates for proteasomal degradation. This figure was created by an author (N.K.J.) using the website https://app.biorender.com [Correction added on 27 April 2021, after first online publication: A typo within Figure 2 has been corrected] FBXW7α upregulates it through a ubiquitin-mediated stabilization. [7][8][9] Overall, the biology of FBXW7 is very complex not only in normal cells but in a wide range of carcinomas as well. It is therefore essential to understand the FBXW7 based interactions with much more clarity to identify disease-critical interactions, which potentially drive pathogenesis in cancer patients.
The effect of E3 ligases on the NOTCH pathway is well known.
Members of several E3 ligase families interact either directly or indirectly with NOTCH and with other NOTCH pathway-associated proteins to facilitate a panel of cellular processes. Therefore, subtle deregulation in the UPS, including the E3 ligases, coupled with NOTCH-based aberrations can disturb normal cellular homeostasis and may drive the cell to a malignant fate. For example, conditional inactivation of Mind bomb-1 (Mib1), a ubiquitin ligase mostly associated with the endocytosis of Delta receptor in the mouse brain, results in the complete loss of NOTCH activity, thereby hampering neural stem cell differentiation. Likewise, Drosophila Su(dx), a member of the homologous to E6-associated protein C terminus (HECT) family of ubiquitin ligases is known to negatively regulate the NOTCH pathway. Even further, the mammalian ortholog of Su(dx), Itch (atrophin-interacting protein 4), has been known to facilitate the polyubiquitination of the membrane-anchored form of NOTCH, leading to its lysosomal degradation. [10][11][12] Several studies have confirmed that the NOTCH intracellular domain (NICD) is the principal target of degradation by the SCF family E3 ligases FBXW7 (Figure 2). An initial study on Sel-10 (nematode homolog of FBXW7) found that NICD is negatively regulated by FBXW7, which results in deactivation of the NOTCH pathway. Over the last decade or so, NOTCH and FBXW7 imperative to understand such disease-critical interactions with a therapeutic perspective in mind. 13

| KEY INTERACTIONS BETWEEN FBXW7 AND NOTCH PATHWAY ASSOCIATED COMPONENTS
Although NICD remains the principal substrate of FBXW7 with respect to the NOTCH pathway, there are emerging evidences that highlight the interaction between FBXW7 and other accessory proteins that are directly or indirectly involved in various key cellular processes and in the discharge of NOTCH signaling duties (Table 1 and Figure 2). This section presents an insight into such novel interactions involving FBXW7 and proteins, such as Presenilins (PS), Hes-1, Hes-5, etc.

| Presenilins
Presenilins (PS) are the catalytic components of the enzyme γ-secretase that is needed for NOTCH pathway activation. It has been suggested that FBXW7α positively regulates the activity of NICD and epidermal growth factor receptor (EGFR), principally by striking a balance between ubiquitylation and stability of both NICD and EGFR. PS also negatively regulates FBXW7 transcriptional activity, thereby T A B L E 1 List of interacting components of FBXW7 and NOTCH and their associated functions in various key cellular processes  18 Angiogenesis FBXW7 and NOTCH FBXW7 is a positive regulator of angiogenesis that restricts the activity of NOTCH in the endothelium of the growing vasculature. 19 Lipid Metabolism FBXW7 and RBP-J FBXW7 plays key roles along with NOTCH cofactor RBP-J in modulating lipogenesis, cell proliferation and differentiation in the liver. 20 Bipotent liver progenitor cells (LPCs) differentiation FBXW7, NOTCH, and Hdac1 Hdac1 regulates the differentiation of LPCs into hepatocytes via Sox9b, and into BECs through Cdk8, FBXW7, and NOTCH3 in zebrafish models. 21 Neural cell differentiation FBXW7, NOTCH, and c-Jun FBXW7 regulates neurogenesis by antagonizing NOTCH and c-Jun Nterminal kinase (JNK)/c-Jun pathway. 22 Osteoporosis FBXW7 and NOTCH Overexpression of NOTCH2 carrying a truncated C terminus that escapes FBXW7-mediated degradation results in sustained osteoclast activity. 23 Neural stem cell fate determination FBXW7, NOTCH, and Hes-5 FBXW7β transcription by NOTCH signaling provides an essential mechanism that is coupled to and absolutely essential for the correct specification of cell fates triggered by lateral inhibition. 24 Cell fate, proliferation, and homeostasis FBXW7, NOTCH, and Alpha-synuclein (SNCA) SNCA promotes degradation of NOTCH1 intracellular domain (NICD) by FBXW7-dependant mechanism, and attenuates the interaction between NICD and RBP-Jk. 25 EGFR signaling and cell transformation FBXW7, NOTCH, and PS PS plays a key role during epidermal growth and transformation by reciprocally modulating the EGFR and NOTCH signaling through FBXW7. 26 positively and negatively controlling EGFR and NOTCH pathway,

| T-cell acute lymphoblastic leukemia
Precursor T-cell acute lymphoblastic leukemia (T-ALL) continues to remain a roadblock in pediatric oncology. T-ALL tumor relapses display extremely poor prognosis, and therefore, it is vital to isolate molecular risk factors, which allow early and efficient treatment strati-  Figure 3 has been corrected] NOTCH1 HD mutations, suggesting that NOTCH pathway activation is one of their major roles in T-cell leukemogenesis. Interestingly, FBXW7 mutations are rarely detected alongside a PEST mutation and it can be conferred that both FBXW7 and PEST mutations increase the stability of the N1-ICD and therefore, may be functionally redundant. 32,33 Increased NOTCH or decreased FBXW7 expression can be asso- Quite astonishingly, the current study found that NOTCH1/FBXW7 MUT and WT groups were equally balanced in terms of percentages of standard-and high-risk patients, thereby implying a more discrete role of mutations in driving prognosis of T-ALL. 36 Lastly, in a few T-ALL cases, mutations have been reported in the IKAROS gene, which can be associated directly or indirectly to NOTCH and/or FBXW7 aberrations. In mouse, T-ALL-activating

| Osteochondromas
Osteochondromas are normally bone protuberances surrounded by a cartilage layer that tends to affect the extremities of the long bones in an immature skeleton and deform them. They normally occur singly but multiple forms of presentation may also be seen in certain cases.
Osteochondromas have a very typical appearance and are easily diagnosed but an atypical site (in the axial skeleton) and/or malignant transformation of the lesion may sometimes make it challenging to identify osteochondromas immediately using radiographic techniques.
There exists a continuing debate on whether osteochondroma is actually a developmental disorder (pseudotumoral lesion) or neoplasm.
Nevertheless, irrespective of whether it is a pseudotumoral lesion or a more common benign bone tumor, it is definitely an exostosis phe- It must be noted that FBXW7 explicitly recognizes phosphorylated substrates. GSK-3β is a key kinase involved in the FBXW7 pathway. There is enough data to suggest that the sequences identified by

| Peptidyl-prolyl cis-trans isomerase
Prolyl-isomerase (Pin1) is the only recognized peptidyl-prolyl cis-trans isomerase (PPIase) that explicitly recognizes and isomerizes the phos- Therefore, therapeutic modalities based on the partial delivery of FBXW7-α to intestinal tumor cells may limit nuclear DEK and β-catenin accumulation and drive better prognosis in patients. 15,65

| Apoptosis
Apoptosis is the mechanism of programmed cell death that is driven downregulation by NOTCH may not be due to decreased transcription or degeneration by caspases but due to enhanced degradation through a FBXW7-dependent process. Collectively, these findings position FBXW7, NOTCH, and MCL1 as key drivers of apoptosis. 68,69

| Metastasis
Metastasis remains a major concern and a principal cause of death in cancer patients. Therefore, elucidation of the genes and mechanisms that control this process has remained an area of active research.
Metastasis essentially involves a complex array of a process that begins with the detachment of cancer cells from a primary tumor followed by their migration to neighboring tissue, entry into the circu-

| Other miscellaneous key oncogenic events
Notch hyperactivation may be caused by mutations of NOTCHassociated genes, altered upstream key oncogenic events, or microenvironment signals. Cancer cells may exploit this aberrant signaling to "educate" the surrounding microenvironment cells toward a pro-tumoral behavior. For instance, in context to tumor microenvironment (TME), it has been observed that epithelial NOTCH1 signaling drives metastasis in serrated colorectal cancer (CRC), where poorprognosis of CRC subtypes CMS4/CRIS-B are controlled by NOTCH1.
Furthermore, TGF-β-mediated neutrophil infiltration has been found critical for NOTCH1-driven metastasis, although neutrophil targeting provides therapeutic opportunity in metastatic CRC. In addition, NOTCH1 signaling can be activated via mutation of FBXW7, found in 11% of human CRCs. Thus, epithelial NOTCH signaling rewires the TME of CRC to drive poor-prognosis subtypes and metastasis. 73 Recently, the role(s) of NOTCH signaling in T-cell antitumor immunity and TCR-and chimeric antigen receptor-based immunotherapies has been reported. NOTCH signaling is required for optimal T-cell- miRNAs have also been implicated in promoting GSI resistance in T-ALL tumors through an interaction with both NOTCH and FBXW7.
Notably, the NOTCH-mediated activation of miR-223 transcription has been found to represses the tumor-suppressive effects of FBXW7 in T-ALL cell lines. miR-223 promoter harbors a conserved RBPjk binding site that is nested within an NF-kB consensus sequence, thereby suggesting that both NOTCH and NF-kB act cooperatively to provide regulatory signals to control miR-223 transcriptional activity.
In line with this observation, NOTCH1, NOTCH3, and p65 have all been found to be directly recruited to the miR-223 promoter site.
Therefore, in T-ALL cells, NF-kB inhibition has been found to limit the expression of endogenous miR-223. Consistent with this, the abrogation of the NF-kB pathway in Jurkat IKKγ−/− cells along with low NOTCH1 and NOTCH3 levels results in the downregulation of miR-223 mRNA levels. Interestingly, ectopic modulation of miR-223 expression was found to limit T-ALL resistance to GSI treatment.
Upregulated miR-223 driven GSI resistance has been attributed in part to FBXW7 and as the data suggest, resistance in Molt3 and Jurkat T-ALL cells may be due to GSI-induced increased C/EBPα expression that leads to a completed loss in FBXW7 activity. Over the last decade, enormous progress has been in the field of cancer therapeutics in addition to the most common and conventional therapies that include surgery, radiation, and chemotherapy. There are drawbacks to these conventional therapeutic protocols, which cause a lot of physical as well as psychological stress among infirm. Chemotherapeutic drugs trigger different types of toxicity in the patient's body, including cardiotoxicity, hematotoxicity, neurotoxicity, gastrointestinal toxicity, hair follicle toxicity, nephrotoxicity, and many more.
Moreover, these drugs target rapidly multiplying cells and are sometime unable to distinguish between normal cells and tumor cells, thereby limiting their maximum allowable doses. On the contrary, these chemotherapeutic agents get swiftly eliminated through the ADME mechanisms, and therefore, administration of a high dosage of the drug is needed to circumvent rapid elimination and for widespread distribution of the drug to the tumor area. Subsequent research advancements have led to the identification of various alternative modalities, such as immunotherapy, liposomal therapy, targeted therapy, hormone therapy, and stem cell therapy, to mitigate the detrimental effects of these drugs. The use of biological molecules to activate the immune system that is severely compromised in patients has been an area of intense deliberation. In contrast to chemotherapy, targeted use of biomolecules is highly efficient against tumor cells and come with limited side effects. Therefore, targeted use of biological and physiological therapies intending to reduce side effects and increasing the long-lasting efficacy will be extremely beneficial in cancer treatment going forward. 89,90 Deregulated FBXW7 and NOTCH interactions can be targeted through novel biomolecules to improve prognosis in tumors. For instance, Chromosome Maintenance Region 1 (CRM1) inhibition that can lead to nuclear retention of tumor suppressor proteins has been explored as a therapeutic strategy in pancreatic ductal adenocarcinoma (PDAC). It was found that CRM1 inhibitors exert their therapeutic effects both in vitro and in vivo through a novel FBXW7 nuclear retention mediated NOTCH1 suppression. CRM1 also known as Exportin 1 (Xpo1) is a protein that has been found to be upregulated in PDAC cells, and blocks the activity of tumor suppressor protein (TSP) through constant nuclear export.
Targeting CRM1 through specific inhibitors of nuclear export ( Table 2   Writing-review and editing.