Hepatitis C

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs

Liver-specific deletion of TBC1D15 attenuates p53 loss

We tested first if hepatocyte-specific TBC1D15 deficiency prevented loss of p53 and liver tumor development by using the inducible Alb::CreERT approach described above (Fig. 1b). We generated NS5A Tg mice carrying a floxed Tbc1d15 locus (Supplementary Fig. 1) which allowed tamoxifen-inducible, hepatocyte-specific ablation of this gene (Alb::CreERT;Tbc1d15f/f;NS5A) (Fig. 1c). Liver tumor incidence in wild type Tbc1d15f/f;NS5A mice after 12 months of alcohol-Western diet feeding was 50%, which was reduced significantly to 16% in TBC1D15-deficient hepatocytes of Alb::CreERT;Tbc1d15f/f;NA5A mice (Fig. 1d). Incidence of liver tumor (left) and tumor pictures (right) of the four genetic groups of mice (Fig. 1d). Mouse tumors have vimentin and AFP expression (Fig. 1f). This genetic manipulation also reduced tumor mass (Fig. 1e) and abrogated NANOG upregulation, phosphorylation of NUMB, and p53 loss in these livers (Fig. 1g). In addition, the percentage of CD133+ TICs in total tumor cells of Alb::CreERT2;Tbc1d15f/f;NA5A mice was decreased by 70% (Fig. 1h, top). These TICs exhibited reduced self-renewal activity in vitro (Fig. 1h, bottom), as compared to CD133+ TICs from the Tbc1d15f/f;NA5A mice. These results underscored the requirement of hepatocyte TBC1D15 for liver tumor development and the generation of TICs in this animal model.

N-terminus of TBC1D15 protein inhibits differentiation

We previously showed that the N-terminal region of TBC1D15 (N-TBC1D15, aa 2 158) containing the 50 aa Canoe homology domain is indispensable for NUMB association and p53 degradation9. We examined if the overexpression of N-TBC1D15 inhibited hepatocyte differentiation and promoted oncogenic transformation of ES cells. This was accomplished using a Cre-activated N-TBC1D15LSL (LSL: Lox-Stop-Lox) expression vector (Fig. 1i) confirmed by immunoblotting (Fig. 1j). Differentiation-induction medium upregulates hepatocyte-specific genes such as Albumin or Hnf4a in ES cells. However, N-TBC1D15 expression inhibited induction of these hepatocyte genes (Fig. 1k). ES cells (with or without expression of N-TBC1D15) were cultured in the hepatocyte differentiation medium, then implanted subcutaneously into NOGTM mice, and tumor development was examined for 60 days. Mice with tumors greater than 25 mm3 were recorded as positive. These N-TBC1D15 overexpressing cells cultured in the differentiation-induction medium prior to transplantation into NSGTM mice formed tumors at an 80% incidence rate. By contrast control cells lacking N-TBC1D15 expression rarely produced tumors (~10%) (Fig. 1l). These results demonstrated that the overexpression of N-TBC1D15 alone is sufficient to suppress hepatocyte differentiation and confer tumorigenic activity in ES cells.

Next, we tested how TBC1D15 regulated NUMB-dependent asymmetric division. CD133+ TICs isolated from the alcohol Western diet-fed Ns5a Tg mice showed symmetric localization of the polarity protein NUMB18,19. This was demonstrated by reduced polarization of fluorescent NUMB, which is indicative of the loss of asymmetric division. TBC1D15 knockdown (KD) in TICs restored cell polarization of NUMB while TBC1D15 overexpression (OE) supported the symmetry of NUMB localization (Fig. 1m and Supplementary Fig. 2c). Silencing of TBC1D15 is correlated with a loss of cell division asymmetry as demonstrated by symmetric cell distribution of LGN (Fig. 1m). TBC1D15 KD in TICs reduced the percentage of TICs showing symmetric division and reciprocally increased asymmetric cell division while TBCD1D15 OE resulted in the opposite effect (Fig. 1n). Nanog KD reduced the percentage of TICs undergoing symmetrical division approaching that observed in mouse hepatoblasts (Fig. 1o). These results validated the pivotal role of TBC1D15 in TIC symmetric cell division.

TBC1D15 interacts with NuMA1 and prevents NuMA1 LGN interaction

While we have shown TBCD1D15 was required for p53 degradation facilitated by NUMB phosphorylation, the mechanism by which TBC1D15 regulates NUMB phosphorylation is unknown. To this end, we searched for in vivo partners of TBC1D15-mediated regulation by large-scale immunoaffinity purification of interacting proteins of endogenous TBC1D15 in TICs and Identification of interaction partners was accomplished by liquid chromatography tandem mass spectrometry (LC MS/MS: Fig. 2a and Supplementary Fig. 2a, b). This approach identified several high-confidence interacting proteins, including NuMA1, NOTCH1/2/3/4, and RANGAP1 (Fig. 2a and Supplementary Figs. 2 and 3). These candidates were also present in the enriched pathway analysis developed from the MS data for TBC1d15 immunoaffinity purified products (Supplementary Fig. 4). Co-IP-Western blot analysis of cell lysates confirmed that TBC1D15 interacted with NuMA1 and RANGAP1 (Fig. 2b). RANGAP1 regulates RAN GTPase activity, expression of AURKA and mitosis20. NuMA1 binds LGN and Dynein to assemble an asymmetric cell division machinery complex. Indeed, 3D computer modeling predicted TBC1D15 would bind NuMA1 (Fig. 2c and Supplementary Fig. 5). Based on the domain section of NuMA, NuMA aa 1788 to the end portion was simulated into the structure. The TBC1D15 of this structure is used to dock with NuMA from 3ro2 with LGN. In silico analysis reveals TBC1D15 binds a domain of NuMA1 that also binds LGN, suggesting a competition between them for binding with NuMA1. TBC1D docked to NUMA with further alignment of LGN (3ro2), showing partial competition for the binding site of NUMA (Fig. 2c, bottom).

Fig. 2: TBC1D15 NuMA1 interaction inhibits asymmetric cell division via inhibition of key asymmetric machinery, NuMA1 LGN interaction.

a Shotgun LC MS/MS was performed on immunoaffinity purified TBC1D15-interacting proteins (highlighted in red boxes; NuMA1, RANGAP1, and NOTCH1, 2, 3, 4). b Co-immunoprecipitation-immunoblot analysis confirmed that TBC1D15 interacts with NOTCH1/2, NuMA1, and RANGAP1. c In silico analysis predicts an interaction between TBC1D15 and NuMA1. (top) Structure model of TBC1D15 (green) binding NuMA1 (blue). (middle) Docking between TBC1D15 and NuMA1 was simulated. (bottom) TBC1D docked to NUMA with further alignment of LGN (3ro2), showing partial competition for the binding site of NUMA. TBC1D15 in green, LGN in blue, and partial NUMA in yellow. d Human TBC1D15 protein has 51% homology with Drosophila cell fate determinant Canoe (SNO: Mammalian AFADIN homologue) that binds mammalian homologue of LGN (Drosophila Pins). e IP-Western blot analysis (left panel) and reverse IP-Western blot analysis (right panel) were performed in TBC1D15 overexpression. f Domain mapping of TBC1D15 binding domain of NuMA1 by using GFP-fused NuMA1 truncation or mutant proteins and Flag-tagged TBC1D15. g The binding of purified flag-TBC1D15 protein with or without purified GFP-NuMA1 mutant protein was tested by in vitro binding assay. h We performed FRET analyses between TBC1D15-CFP and NuMA1-YFP. (left upper panel) Cartoon depicting binding partners TBC1D15 and NuMA1 tagged with fluorescent proteins CFP and YFP. (left bottom panel) Living CD133+ cells transfected with the controls CFP only, YFP only, CFP and YFP as well as the CFP YFP fusion proteins were analyzed on a FACS flow cytometer. (right panel) Representative optical images showed CFP and YFP expression in CD133+ cells with CFP and YFP. CFP is shown in red and YFP in green (Supplementary Fig. 10). i Hypothetical model of TBC1D15-mediated disruption of the interaction between NuMA and LGN that inhibits asymmetric cell division and promotes symmetric cell division. Source data are provided as a Source Data file.

From in silico structural simulations of NuMA1, T1804 was more important than S1969 of NuMA1 C-terminal fragment for mediating an interaction with the N-terminal domain (aa 1 270) of TBC1D15. The G168 residue of TBC1D15 (green) interacted with T1804 of NUMA (blue) (Fig. 2c, bottom). Human TBC1D15 protein has 51% homology to Drosophila cell fate determinant Canoe (CNO) (Fig. 2d) that binds mammalian homologue of Pin (LGN)21. Furthermore, NuMA1 interaction with LGN was suppressed by TBC1D15 as shown by co-IP-Western blot analysis (Fig. 2e, left panel) and reverse co-IP-Western blot analysis (Fig. 2e, right panel). These results demonstrate that TBC1D15 NuMA1 interaction potentially prevents interaction between NuMA1 and LGN. These results indicated that TBC1D15 interacts with NuMA1 to prevent NuMA1 LGN interaction (Fig. 2e).

To map a TBC1D15 interaction site of NuMA1, we generated GFP-fused NuMA1 proteins with truncations and mutations to test their interactions with Flag-tagged TBC1D15 (Fig. 2f). The GFP-NuMA1 C-terminus (aa 1700 2115), but not the N-terminus (aa 1 213) interacted with TBC1D15. This C-terminal region contains S/T residues including serine or threonine residue which are phosphorylated by AURKA resulting in dissociation from LGN and a loss of cell division symmetry22. Alanine substitutions of three such residues (GFP-NuMA1 C-3A) largely abrogated the NuMA1 interaction with TBC1D15, suggesting the importance of serine or threonine phosphorylation for TBC1D15’s interaction with the NuMA1 C-terminus for disrupting asymmetric cell division control.

We performed new experiments and have added the in vitro binding assay of TBC1D15 and NuMA1 (wild-type or T1804A, S2047A, and T1991A mutation). Actually, in this manuscript, we tested a single alanine substitution of T1804 (NuMA1 T1804A). And we suggested that the importance of T1804 phosphorylation for TBC1D15’s interaction with the NuMA1 C-terminus for disrupting asymmetric cell division control in Fig. 2i. We examined alanine substitutions of S2047A, T1991A, and T1804 (Fig. 2g).

To measure FRET signals by FACS, CD133 positive cells expressing either the cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP) were analyzed (Fig. 2h, top). Compared to cells transfected with 40% positive control cells transfected with a CFP YFP fusion protein, only 10% of cells co-transfected with CFP and YFP were recorded as FRET positive (Fig. 2h, right).

We speculated that the TBC1D15 NuMA1 interaction potentially prevents interaction between NuMA1 and LGN, thus leading to inhibition of asymmetric cell division (Fig. 2j, left). This model TBC1D15 structure was used to dock to the NuMA1 LGN complex (Fig. 2j, right). Indeed, this 3D conformational modeling predicted that TBC1D15 would partially occupy the LGN position with NuMA1 (PDB 3ro2) (Supplementary Fig. 5, right).

Translational relevance of TBC1D15

We sought to determine the translational relevance of the tumor promotion activity of TBC1D15 as described in the present studies. For this, the TCGA liver cancer data were analyzed for expression of TBC1D15 and NOTCH1 in relation to HCC stage. These data showed co-overexpression of these two proteins was highest in the late metastatic stage as compared to normal or earlier stages of HCC, suggesting a role for the tumor-promoting TBCD1D15 NOTCH interaction (Fig. 3a and Supplementary Table 1 for Box plot statistics). Based on this meta-analysis we examined the expression of NANOG, TBC1D15, p53, N1ICD, and p-(S265) NUMB in liver protein extracts of normal human subjects vs patients with alcoholic cirrhosis/hepatitis (Fig. 3b, c). Hypothetical model of TBC1D15-mediated NOTCH activation and stabilization leading to NICD-dependent transactivation of the Nanog gene to generate TICs, leading to HCC development. Normal liver progenitor cells (LPC) differentiate into either cholangiocytes or hepatocytes through NOTCH signaling or NUMB-mediated signaling, respectively (Fig. 3c). This expression signature was associated with a high propensity for liver cancer development (Fig. 3b). The results clearly demonstrated the coordinated upregulation of NANOG, TBC1D15, N1ICD, and p-(S265) NUMB with p53 depletion in these patients. From this analysis, we concluded that TBC1D15 was an important mediator in two oncogenic pathways (loss of p53 and NOTCH activation).

Fig. 3: TBC1D15 NOTCH1 interaction promotes self-renewal and oncogenesis.

a Late stage HCC patients exhibit higher NOTCH1 and TBC1D15 combined expression (TCGA): Each circle represents the expression of a single sample (p < 0.0001). b Explant liver tissues from alcoholic cirrhosis/hepatitis patients (immunoblots). c Hypothetical model. d Co-IP-Western blot analyses confirmed the interaction of TBC1D15 with full-length NOTCH1/2/3/4. e Immunoblot analyses performed expression of NOCH1 and N1ICD in TICs and primary hepatocytes. f CD133+ Huh7 cells express a higher level of N1ICD compared to CD133− cells (immunoblot). g TBC1D15 overexpression elevated N1ICD in PH5CH cells (immunoblot). h Colony formation (left) and expression of Nanog mRNA (right) in TBC1D15, sh-Scramble, and sh-NOTCH expressing PIL4 cells. Colony numbers were calculated as mean ± SD (n = 3). [left; *p = 0.00145 (TBC1D15 vs sh-NOTCH); **p = 0.0056 (Vector vs TBC1D15); **p = 0.01668 (sh-Scramble vs sh-NOTCH); Student’s t test]. p-Values by two-tailed paired t test. [right; *p = 0.004988 (TBC1D15 vs sh-NOTCH); **p = 0.002575 (Vector vs TBC1D15); **p = 0.02014 (sh-Scramble vs sh-NOTCH); Student’s t test]. i TBC1D15 KD abrogated the activity of HEY1 promoter-luciferase in Huh7 cells. Data are represented as ±SD (n = 4). p-Values by two-tailed paired t test. *p = 0.00953 (shTBC1D15 vs sh-Scramble; Hey1); *p = 0.018604 (sh-TBC1D15 vs sh-Scramble; Δ215); Student’s t test. j CSL-mediated Nanog activation. k Six mutant-luciferase constructs. Data are represented as ±SD (n = 3). p-Values by two-tailed paired t test. *p = 0.008879 (WT vs M1); *p = 0.000117 (WT vs M2); *p = 0.000262 (WT vs M3) (Student’s t test). l TBC1D15 or NOTCH1 expression promotes hepatoblast proliferation. Data are represented as ±SD (n = 4). p-Values by two-tailed paired t test. *p = 0.0001149 (Vector vs TBC1D15); **p = 0.00001853 (Vector vs NOTCH1; Student’s t test). mTBC1D15 KD reduced N1ICD but increased NOTCH1 in Huh7 cells. m CD133+ Huh7 cell lysates were analyzed for immunoblots in the presence of shRNA targeting TBC1D15 or scrambled shRNA. TBC1D15 KD reduced N1ICD but increased NOTCH1 in Huh7 cells. n Tumor growth after TIC transplantation in NSGTM mice is suppressed by TBC1D15 KD. Error bars represent ±SEM. o Tumor volume determined 2 months after transplantation of TICs into NSGTM mice. Error bars represent ±SEM (n = 4). p-Values by two-tailed unpaired t test. *p = 0.00000233 (NICD vs shTBC1D15); **p = 0.0000181 (sh-Scramble vs NIICD) (Student’s t test). p TBC1D15 KD reduced NOTCH1 stability. NOTCH1 protein was immunoprecipitated and detected by immunoblotting (left). The NOTCH1 autoradiographic reading (right). Source data are provided as a Source Data file.

TBC1D15 interacts with NOTCH to activate NOTCH pathway

Our immunoaffinity purification for TBC1D15 interacting proteins, identified NOTCH1, 2, 3, and 4 as high-confidence interacting proteins (Supplementary Fig. 4). Co-IP-Western blot analysis of TIC cell lysates confirmed TBC1D15 interacted with all NOTCH isoforms in both full length and activated NICD forms (Fig. 3d). TICs expressed cleaved NICD while primary hepatocytes expressed full-length NOTCH1 (Fig. 3e). Sorted CD133+ Huh7 cells also expressed activated NOTCH 1 (NICD) while CD133− cells only expressed the full-length NOTCH1 (Fig. 3f). This suggested NOTCH pathway activation occurred in tumorigenic CD133+ cells. To test the effects of TBC1D15 OE on full length NOTCH1 and N1ICD levels, we used non-transformed cell line PH5CH, which has no or minimal N1ICD (Fig. 3g, lane 2). TBC1D15 OE resulted in increased N1ICD (Fig. 3g, lane 1). To dissociate the effect of NOTCH1 activation, we treated the cells with the γ-secretase inhibitor DAPT and repeated the same TBC1D15 OE in PH5CH cells. Under this condition, both full-length NOTCH1 and N1ICD were evidently increased (Fig. 3g, lane 3), consistent with TBC1D15 stabilization of NOTCH1/N1ICD and/or activation of NOTCH1 in a manner independent of γ-secretase23. DAPT, an inhibitor of the γ-secretase complex, indirectly inhibits the pathway of Notch, a key target of γ-secretase. A γ-secretase inhibitor prevents proteolytic cleavage of the Notch intracellular domain (NICD), which is necessary for the Notch signaling response when NICD translocate to the nucleus and affect transcription24,25.

Although we demonstrated TBCD1D15-facilitated p53 degradation and TIC self-renewal in other liver cell lines, we noticed that the loss of p53 occurred only in hepatoblasts (PIL4 cells) and failed to result in transformation. Transformation independent of p53 was observed when TBC1D15 was expressed in these cells leading to increased self-renewal (colony formation) (Fig. 3h, left) and higher NANOG expression (Fig. 3h, right). We tested if these effects required the NOTCH pathway. Indeed, NOTCH1 KD significantly attenuated the observed transformation effects of TBCD1D15 in these cells (Fig. 3h). To examine the functional relationship of the NOTCH pathway to TBC1D15, we knocked down TBC1D15 (TBC1D15 KD) in Huh7 cells. This manipulation caused a loss of HEY1-promoter activity, the conventional readout of NICD transactivity (Fig. 3i), indicating that TBC1D15 enhances activation of a NOTCH target gene.

Novel CSL sites in Nanog gene

We have previously shown that TIC self-renewal and tumorigenic activity require NANOG which is transcriptionally induced by TLR4 signaling in part via E2F17, However, even without LPS-TLR4 stimulation, TICs maintain the self-renewal phenotype, indicating that TICs possess an intrinsic mechanism to promote transcription of this stemness gene. Using the bioinformatics tools, we have identified a previously unreported CSL/RBPJ site for binding NICD in the proximity of the OCT4/SOX2 binding element of Nanog promoter (−212/−119) (Fig. 3j). Additionally, a functionally important E2F1 binding site is found within the distal enhancer, 5 kb upstream of the transcription initiation site (TSS) of Nanog gene7,26. Just downstream of this E2F site, we have identified a second CSL/RBPJ sequence element (see Fig. 3j).

We characterized the functionality of these two putative CSL sites in Nanog for transcriptional activity. Two regions together may account for majority of the reporter activity, including the enhancer region (−5421/−4828) containing the E2F1 and CSL elements and the TSS proximal promoter region (−153/+1) containing the CSL and OCT4/SOX2 sites7. Mutations in CSL elements reduced Nanog promoter luciferase activities, indicating that CSL binding sites in these regions are critical for Nanog promoter activities.

The overexpression of TBC1D15 or NOTCH1 increases hepatoblast proliferation (Fig. 3l). Freshly-isolated TICs transduced with shRNA lentivector were transplanted subcutaneously into NSG mice forming a growing tumor which was reduced in size by more than 80% upon TBC1D15 KD (Fig. 3m, n). Concomitant expression of Notch 1 intracellular domain (N1ICD), partially rescued this inhibition of tumor growth (Fig. 3n). Conversely, N1ICD expression alone resulted in a significant increase in tumor growth promotion that was attenuated by TBC1D15 KO (Fig. 3n) to the level below the growth achieved in control TICs (Fig. 3n, o). These results indicated TBC1D15 has NICD-dependent and -independent tumor promoter activities, and that the NICD-dependent effect is at a level downstream of NOTCH activation.

We examined the cooperative relationship between TBC1D15 NOTCH/N1ICD in a more physiologic context. PIL4 hepatoblasts with or without lentivirus-based N1ICD expression and/or Tbc1d15 KD were orthotopically transplanted into the left liver lobe of NSG mice. PIL4 cells expressing N1ICD expression formed large tumors within 2 months of transplantation, but TBC1D15 KD in these cells significantly reduced this growth, confirming that the N1ICD tumor promotion effect required TBC1D15 (Fig. 3n, o). These results were consistent with the model that TBC1D15 rendered tumorigenic effects partly via its ability to activate NOTCH and to support N1ICD activity. In support of this mode of NOTCH activation, TBC1D15 knockdown in Huh7 cells diminished N1ICD levels in favor of increased levels of full-length NOTCH-1 (Fig. 3m).

A mechanism by which TBC1D15 supports NICD activity is by stabilization of N1ICD. To test this possibility, we examined N1ICD protein stability in Huh7 cells stably transfected with shRNA for TBC1D15 vs scrambled shRNA. Cells were radiolabeled with 35S-methionine and whole cell lysates were prepared at timed intervals following cycloheximide treatment. This analysis showed that labeled N1ICD protein was detected in cells when transduced with scrambled shRNA with a half-life of 162 min (Fig. 3p). By contrast, the cells treated with sh-TBC1D15, mainly showed labeled full-length NOTCH-1 but very little N1ICD that was more rapidly degraded with a half-life of 88 min. These results indicated that TBC1D15 promoted NOTCH-1/N1ICD stability by specific TBC1D15 NOTCH interaction.

NOTCH1 TBC1D15 interaction domains

We next performed domain mapping studies to define a TBC1D15 interaction site of NOTCH1 by using deletion mutants (Fig. 4a). We tested the ability of wild-type NOTCH1 or a constitutively stable NOTCH1 mutant (PEST domain of NOTCH 1 C-terminal). These mutants lack the constituting the consensus for the E3 ubiquitin-ligase FBW7A, the major negative regulator of the intracellular NOTCH signal27,28. This analysis demonstrated that TBC1D15 interacted with NOTCH1/N1ICD within a region spanning aa 2171 2473 of the C-terminal PEST domain known for the initiation of protein degradation29 (Fig. 4a, b). Note Myc-tagged TBC1D15 interacted with intact N1ICD-HA and N1ICD with a partial deletion of the PEST domain distal to aa 2473: N1ICDΔPEST (aa 1754 2473) but not with N1ICD with a complete deletion of the PEST domain: N1ICDΔPEST (aa 1754 2171) (Fig. 4b). In fact, NUMB is known to bind this PEST domain to recruit the E3 ubiquitin ligases, ITCH and FBW7 for ubiquitination and subsequent degradation of NOTCH1 and N1ICD (Supplementary Fig. 8)30. As TBC1D15 interacts with NUMB, our results suggested that TBC1D15 interfered with NUMB-mediated NOTCH/N1ICD degradation by binding to the same PEST domain.

Fig. 4: NOTCH1 C-terminal PEST domain interacts with N-terminus of TBC1D15 for cooperative oncogenic activity.

a Scheme representation of NOTCH-1 deletion mutants were generated. Co-IP-Western blot analysis identified a region between aa 2171 and aa 2473 of the PEST domain as the interaction site. b Co-IP-Western blot analysis identified interaction domains of TBC1D15 interacted with intact N1ICD-HA and N1ICDΔPEST (aa 1754 2473). c The N-terminus but not C-terminus of TBC1D15 interacts with NOTCH-1. A similar domain mapping of TBC1D15 for its interaction with NOTCH-1 revealed aa 1 200 N-terminal fragment with known lysine residues for ubiquitination (Myc-HA-TBC1D15-N term) but not the C-terminus (Myc-HA-TBC1D15-C term), interacted with NOTCH-1. Substitution of these lysines with arginine (Myc-HA-TBC1D15-N 2KR) did not affect the binding. d Co-IP/IB showed TBC1D15 NOTCH1 interaction was lost by deletion of the N-terminus interacting domain (ΔN) while the TBC1D15 NUMB interaction was lost by deletion of the CNO domain (ΔCNO). NICD expression was lost when TBC1D15 with ΔN was expressed while p53 was restored when TBC1D15 with ΔCNO was expressed. This underscored the importance of these distinct domains for the two respective functions. e Additional deletion analysis revealed the N-terminal domain spanning aa.163 217 upstream of the NUMB-binding Canoe (CNO) domain (aa.218 270) was responsible for TBC1D15 interaction with NOTCH. f ΔCNO, ΔN, or both domains of TBC1D15 was reduced spheroid formation of PH5CH cells. Spheroid numbers were counted as mean ± SD (n = 4). p-Values by two-tailed unpaired t test. **p = 0.0001544 (Vector vs TBC1D15); *p = 0.0002660 (TBC1D15 vs ΔCNO); *p = 0.00008665 (TBC1D15 vs ΔN); *p = 0.00035214 (ΔCNO vs ΔN) (Student’s t test). g ΔCNO, ΔN, or both domains incrementally contributed to the abrogation of TBC1D15-induced tumor formation (tumor pictures; left/middle and tumor volumes; right) in NSGTM mice. The visible tumors were measured at the indicated days, Error bars represent ±SD (n = 4). p-Values by two-tailed unpaired t test. **p = 0.00001448 (Vector vs TBC1D15); *p = 0.00003475 (TBC1D15 vs ΔCNO); *p = 0.00001831 (TBC1D15 vs ΔN); *p = 0.00009508 (ΔCNO vs ΔN) (Student’s t test). Source data are provided as a Source Data file.

We performed the reciprocal structure mapping of TBC1D15 to identify its interacting domain with NOTCH/NICD. The N-terminal (aa 1 200) but not a C-terminal fragment of TBC1D15 was found to bind NOTCH1 (Fig. 4c). The TBC1D15 N-terminal fragment has two lysine residues identified as ubiquitination sites for the initiation of degradation9. A degradation-resistant mutant with these two lysine residues replaced with arginine (myc-HA-TBC1D15-N-2KR), also interacted with NOTCH-1 (Fig. 4c). Further deletion analysis of TBC1D15 within the N-terminus revealed a domain spanning aa 218 270 distinct from the NUMB-binding Canoe (CNO) domain (aa 163 217) which interacted with NOTCH/NICD (schematically shown in Fig. 4e). The latter result also showed that N1ICD interaction was lost when TBC1D15 lacking the NOTCH interacting domain (TBC1D15ΔN or TBC1D15ΔCNOΔN) was expressed and assayed in cell culture, whereas the p53 level was restored when TBC1D15 lacking the CNO domain was expressed (either TBC1D15ΔCNO or TBC1D15ΔCNOΔN) was expressed. Binding of NUMB to this TBC1D15 region was corroborated by co-IP results shown in Fig. 4d. These results confirmed both CNO and NOTCH1 binding domains of TBC1D15 are important for N1ICD expression and p53 turnover.

Functional significance of the NOTCH interacting and CNO domains of TBC1D15 was shown by incrementally deletions of TBC1D15 and corresponding reduced spheroid formation (Fig. 4f) or tumor growth of non-neoplastic immortalized human cell line, PH5CH31 in NSG mice (Fig. 4g). By deletion of both NUMB-interacting CNO domain and NOTCH-interacting N-terminal domain in TBC1D15ΔCNOΔN (with possible dominant negative phenotype) we observed maximum suppression of tumor formation, highlighting the importance of these two domains in vivo (Fig. 4g).

RANGAP1 TBC1D15 association promoted NUMB phosphorylation

NUMB is phosphorylated at S284 and S265 contributing to the loss of cell division asymmetry20. TBC1D15 KD or OE respectively suppressed or increased S265 phosphorylation in Huh7 cells (Fig. 5a), and these effects correlated with TBC1D15’s ability to regulate aPKCζ activity (Fig. 5c). However, TBC1D15 KD did not affect protein levels of various kinases that may potentially phosphorylate NUMB, including aPKCζ (Fig. 5b, c). The ability of TBC1D15’ ability to interact with RANGAP1 suggested that RANGAP1 may positively regulate AURKA and microtubule kinetochore interaction as previously suggested13, which in turn activates aPKCζ for NUMB phosphorylation. This notion was tested by cell-free kinase assay using Huh7 cell lysate and NUMB as a substrate (Fig. 5d). NUMB phosphorylation was increased by recombinant aPKCζ (raPKCζ) in a dose-dependent manner. NUMB was dose-dependently phosphorylated by recombinant aPKCζ at concentrations up to 1 μM (Fig. 5d(i)). TBC1D15 enhanced NUMB phosphorylation mediated by aPKCζ and AURKA, and this effect was lost when AURKA is absent. Vector-mediated expression of aPKCζ with or without AURKA expression, only marginally increased NUMB phosphorylation, while concomitant expression of TBC1D15 with AURKA and aPKCζ, conspicuously increased the NUMB phosphorylation. This effect was lost without AURKA expression (Fig. 5d(ii)), supporting the critical role of TBC1D15 AURKA cooperation in aPKCζ-mediated NUMB phosphorylation. The AURKA inhibitor MK5108 abrogated the TBC1D15 AURKA aPKCζ cooperative NUMB phosphorylation. In fact, the AURKA inhibitor MK5108 completely abrogated the phosphorylation by this cooperative action of TBC1D15 AURKA aPKCζ (Fig. 5d(iii)). This line of regulation was investigated further. We then tested the effect of RANGAP1 expression on the phosphorylation achieved by TBC1D15, AURKA, and aPKCζ. RANGAP1 OE indeed rendered incremental and dose-dependent upregulation of the phosphorylation achieved by TBC1D15 AURKA aPKCζ (Fig. 5d(iv)). Conversely, lentiviral RANGAP1 KD by RANGAP1 shRNAs in Huh7 cells prior to in vitro kinase assay, significantly reduced NUMB phosphorylation (Fig. 5d(v)).

Fig. 5: TBC1D15 promotes NUMB phosphorylation through RANGAP1-mediated promotion of AURKA aPKCζ activation.

a TBC1D15 knockdown inhibits NUMB phosphorylation at S265 as demonstrated by immunoblot analysis. b TBC1D15 knockdown or overexpression does not affect kinase protein levels as demonstrated by immunoblot analysis. c TBC1D15 knockdown reduces and TBC1D15 overexpression increases aPKCζ kinase activity demonstrated aby in vitro kinase assays. Data are represented as ±SD (n = 4). p-Values by two-tailed unpaired t test. *p = 0.00027596 (shTBC1D15 vs shScramble), *p = 0.000008157 (shScramble vs TBC1D15 OE) (Student’s t test). d In vitro cell-free NUMB phosphorylation assay. Each bar represents the mean ± SD of four independent experiments. p-Values by two-tailed unpaired t test. *p = 0.000139 (TBC1D15 AURKA aPKCζ-NUMB vs AURKA aPKCζ-NUMB), *p = 0.0002468 (TBC1D15 AURKA aPKCζ-NUMB vs TBC1D15-aPKCζ-NUMB), *p = 0.00000058 (TBC1D15 AURKA aPKCζ-NUMB vs TBC1D15 AURKA aPKCζ-NUMB-0.25μg RANGAP1), *p = 0.00008243 (TBC1D15 AURKA aPKCζ-NUMB vs TBC1D15 AURKA aPKCζ-NUMB-0.5 μg RANGAP1), *p = 0.0007145 (TBC1D15 AURKA aPKCζ-NUMB-shScramble vs TBC1D15 AURKA aPKCζ-NUMB-shRANGAP1) (Student’s t test). e (top) RANGAP1 knockdown abrogated and RANGAP1 overexpression (OE) enhances AURKA interaction with aPKCζ and TBC1D15 in Huh7 cells as determined by co-IP-Western blot analysis. (bottom) Immunoblot of total cell lysate reveals p-NUMB was reduced by RANGAP1 KD and increased by RANGAP1 OE. PAR6. f We tested if serine or threonine residue of NuMA1 is phosphorylated by Aurora-A, and that this phosphorylation is important for association with TBC1D15. Serine or threonine residue of NuMA1 is phosphorylated by Aurora-A. g Phosphorylation of serine or threonine residue of NuMA1 by Aurora-A is important for asymmetric cell division, judged by immunofluorescence staining of α-Tubulin and NuMA1-GFP intensity. Bar graph represents fold enrichment of GFP-NuMA1. Scale bar, 10 μm. Data are represented as ±SD (n = 3). p-Values by two-tailed paired t test. *p = 0.000667 (WT vs T1991A) *p = 0.000936 (WT vs S2047A) (Student’s t test). h Hypothetical model that TBC1D15 expression promotes NUMB phosphorylation via RANGAP1 TBC1D15 interaction enhancing AURKA aPKCζ interaction and activation. Source data are provided as a Source Data file.

To gain mechanistic insights into the observed effects of RANGAP1, we tested if RANGAP1 KD or OE influenced AURKA aPKC association. Co-IP-Western blot demonstrated that RANGAP1 KD significantly reduced AURKA aPKC and AURKA TBC1D15 associations while RANGAP1 OE promoted both associations (Fig. 5e, top). Parallel immunoblotting also showed RANGAP1 KD reduced p-NUMB (Ser265) while RANGAP1 OE significantly increased it (Fig. 5e, bottom), indicating that RANGAP1 TBC1D15 association promoted NUMB phosphorylation by increased interactions among TBC1D15, AURKA, and aPKCζ (Fig. 5f). We tested if serine or threonine residue of NuMA1 is phosphorylated by Aurora-A, and that this phosphorylation is important for association with TBC1D15. Serine or threonine residue of NuMA1 is phosphorylated by Aurora-A. Phosphorylation of serine or threonine residue of NuMA1 by Aurora-A is important for asymmetric cell division, judged by immunofluorescence staining of α-Tubulin and NuMA1-GFP intensity (Fig. 5f). The spindle pole in NuMA1 WT vs mutant displayed different degree of NuMA1-GFP intensity. Bar graph represents fold enrichment of GFP-NuMA1. GFP fluorescence intensity was compared in NuMA1 WT vs mutants (Fig. 5g). These results indicate that TBC1D15 RANGAP1 interaction activated NUMB phosphorylation via TBC1D15 AURKA aPKCζ-association (Fig. 5h).

Non-phosphorylatable NUMB mutant prevents oncogenesis

We next tested the contributory role of NUMB phosphorylation in the genesis of TICs and liver tumors in an HCV Ns5aTg mouse model5 fed with an alcohol Western diet. Our previous study with NUMB site-directed mutants demonstrates that the triple substitution-mutant of NUMB-3A (carrying non-phosphorylatable SA substitutions at S7, S265, S284; see Fig. 6a) stably binds p53, while the NUMB-3D (SD phosphomimetic substitutions at these residues) does not12. We used the CRISPR-Cas9 system (Supplementary Figs. 6, 7) to generate transgenic mice to conditionally express in transgenic mice this non-phosphorylatable-mutant NUMB-3A (NUMB-3A) with HA-epitope tag (Supplementary Fig. 6d). These mice were used for further tumorigenesis studies. This approach enabled a tightly controlled analysis of NUMB-3A mediated effects on the genesis of TICs and liver tumors in Alb::CreERT2;Numb3ALSL;Ns5a vs Numb-3ALSL;Ns5a mice (Fig. 6b). Under normal chow feeding, none of the transgenic mice developed liver tumors, including Ns5a Tg mice. The alcohol-Western diet resulted in a liver tumor incidence of 10% in all transgenic mice strains. NUMB3A expression was notable in that liver incidence was reduced to 5%. By contrast normal chow feeding of these animals did not result in tumor formation (Fig. 6c, left). By comparison, in Numb-3ALSL;Ns5a mice (without NUMB-3A expression) fed alcohol-Western diet, a 54% incidence of liver tumors was observed after 12 months of feeding. Histological analyses showed that these mice developed HCC (Fig. 6c, bottom) that were positive for Vimentin and AFP (Fig. 6c, bottom right).

Fig. 6: Targeted expression of a non-phosphorylatable mutant of NUMB reduces HCC development induced by alcohol-HCV synergism mouse model.

a Non-phosphorylatable mutant of NUMB (named as NUMB-3A) was generated by serine-to-alanine substitutions of three NUMB phosphorylation sites. b The experimental strategy of tamoxifen-inducible Cre-mediated NUMB-3A expression using Alb::CreERT2; NUMB3ALSL;NS5A mice. c Incidence of liver tumor (upper left panel), ratio of tumor mass/liver weight (%) (upper middle panel) and liver tumor pictures (upper panel, right) of the four groups of mice was decreased in Alb::CreERT2; NUMB3ALSL;NS5A mice with Ethanol WD. The visible tumors were measured at the indicated days. Error bars represent ±SEM (n = 3). p Values are shown from a chi-square test *p = 0.0003773 (chi-square test). H&E staining for mouse tumors (bottom left panel). Immunofluorescence staining of Vimentin and AFP in tumor tissues (bottom right panel). Scale bar, 30.32 μm. The representative pictures are shown from three independent experiments. d (top) The percentage of CD133+ TICs in tumor cells is compared among the four different genotypes of mice. (bottom) Tumor volume kinetics of TIC-derived tumors. The percentage of TICs of tumor cells were calculated as mean ± SD (n = 3). p-Values by two-tailed paired t test. *p = 0.022474 (Student’s t test). e (left) Immunoblots of tumor cell lysates isolated from hepatocyte-specific expression of NUMB-3A mice. (right) Schematic presentations of LPS-activated TLR4 inducing NANOG mediated NUMB phosphorylation for p53 loss and TIC self-renewal, and of NUMB-3A-antagonism of this pathway. f Micro-CT imaging and texture-based VRT were reduced tumor size and tumor volume. The bar graph shows changes in tumor volume before and after the aPKCζ inhibitor. Error bars represent ±SEM. p-Values by two-tailed paired t test. *p = 0.00654 (aPKCζ inhibitor; before vs after treatment), *p = 0.00095 (after treatment; vehicle vs aPKCζ inhibitor) (Student’s t test). g Τhe aPKCζ inhibitor treatment reduced liver tumors spontaneously developed in HCV NS5A Tg mice fed alcohol Western diet as shown by ultrasound sonography. h Immunoblots of tumor cell lysates isolated from NS5A Tg mice fed alcohol WD of the aPKCζ inhibitor vs vehicle. i Summary of oncogenic pathways. NUMB phosphorylation and TBC1D15 are mutually required for liver tumorigenesis. Source data are provided as a Source Data file.

The role of NUMB3 was illustrated by Alb::CreERT2;Numb3ALSL;Ns5a mice fed the same diet but expressing the mutant NUMB3A. These mice exhibited a significantly reduced tumor incidence of 15%. NUMB-3A overexpression also reduced the average tumor mass by 65% in these mice compared to Numb-3ALSL;Ns5a mice (Fig. 6c, right). Concomitantly, the percentage of FACS-sorted CD133+CD49f+ TICs decreased by 88% in tumors of Alb::CreERT2;Numb3ALSL;Ns5a mice (Fig. 6d, top) compared to Numb-3ALSL;Ns5a mice. Furthermore, the TICs isolated from the former mice exhibited decreased tumor-initiating activity compared to the cells from the latter after transplantation into immunocompromised, recipient NSG mice (Fig. 6d, bottom). Hepatocyte-specific expression of the mutant NUMB-3A in these mice was confirmed by immunodetection which was accompanied by the prevention of p53 loss and NANOG upregulation with attenuated TBC1D15 induction (Fig. 6e, left). These results indicated that tissue-specific (hepatocyte) NUMB phosphorylation indeed contributed to p53 loss and liver tumor development but also resulted in NANOG and TBC1D15 upregulation, suggesting a feed-forward mode of regulation (Fig. 6e, right).

Decoy peptide of NUMB phosphorylation reduces tumor size

As a complementary approach, tumor-bearing Ns5a Tg mice were fed alcohol-Western diet for 10 months, treated with an aPKCζ inhibitor for 4 weeks and examined for effect by live animal imaging. The inhibitor used to antagonize NUMB phosphorylation is a membrane-permeant, synthetic pseudo-substrate, synthetic peptide, myristoyl-SIYRRGARRWRKL32,33. As a control, the inhibitor-free vehicle was administered. The aPKC-ζ pseudosubstrate peptide was injected i.p. into the tumor-bearing NS5A Tg mice (50 μmol/mouse, daily, 5 times per week for 4 weeks). Contrast-enhanced microCT and texture-based volume renderings (VRT) were performed to demonstrate a reduction of tumor size and to measure tumor volume based on 3D data collected (Fig. 6f). This treatment significantly reduced tumor size and volume compared to the vehicle treatment, as assessed by ultrasound sonography and micro-CT imaging (Fig. 6f, g); thus, this demonstrated the in vivo efficacy of the aPKCζ inhibitor in the context of NUMB activity.

Furthermore, immunoblotting of tumor tissues showed marked suppression of NUMB phosphorylation and p53 upregulation, attesting to the efficacy of the aPKCζ inhibitor for prevention of p-NUMB and p53 loss (Fig. 6h) as the most likely basis of tumor suppression. This treatment also suppressed NANOG in concordance with the positive feedback loop shown above. Treatment with the aPKCζ pseudo-substrate inhibitor in HCV NS5A Tg mice-fed AWD for 11 months resulted in >85% reduction in tumor size within 4 weeks, as assessed by micro CT examination of liver tumors.

In summation, our research described the molecular mechanisms of the oncogenic activity of TBC1D15 overexpressed in tumor cells and TICs. TBC1D15 has two major tumor-promoting pathways: interference of the asymmetric division machinery by interacting with NuMA1 and disruption of NuMA1 LBN association. The latter is essential for asymmetric division, and interaction with RANGAP1, leading to AURKA aPKCζ-induced NUMB phosphorylation and p53 degradation (Fig. 6i and Supplementary Fig. 8). This pathway involves TBC1D15 interaction with NOTCH/NICD resulting in NOTCH activation and stabilization. Based on this mechanistic information, we identified a small chemical inhibitor which blocked the TBC1D15 NOTCH/NICD interaction and successfully used it to prevent HCC growth in the PDX in vivo model. Lastly, the prevention of natural liver tumor formation by hepatocyte-specific TBC1D15 deficiency or NUMB mutations in NS5A Tg mice fed alcohol western diet, validated the importance of these two gene products in liver oncogenesis.


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