NUMB and NUMBL differences in gene regulation

Oncotarget9219 www.impactjournals.com/oncotarget NUMB and NUMBL differences in gene regulation José Manuel García-Heredia1,2,3 and Amancio Carnero1,3 1Instituto de Biomedicina de Sevilla, IBIS, Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas, Seville, Spain 2Department of Vegetal Biochemistry and Molecular Biology, University of Seville, Seville, Spain 3CIBER de Cáncer, Instituto de Salud Carlos III, Pabellón 11, Planta 0, Madrid, Spain Correspondence to: Amancio Carnero, email: [email protected] Keywords: NUMB; NUMBL; cancer; Notch pathway; WNT pathway Received: September 06, 2017 Accepted: January 03, 2018 Published: January 11, 2018 Copyright: García-Heredia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT NUMB, and its close homologue NUMBL, behave as tumor suppressor genes by regulating the Notch pathway. The downregulation of these genes in tumors is common, allowing aberrant Notch pathway activation and tumor progression. However, some known differences between NUMB and NUMBL have raised unanswered questions regarding the redundancy and/or combined regulation of the Notch pathway by these genes during the tumorigenic process. We have found that NUMB and NUMBL exhibit mutual exclusivity in human tumors, suggesting that the associated tumor suppressor role is regulated by only one of the two proteins in a specific cell, avoiding duplicate signaling and simplifying the regulatory network. We have also found differences in gene expression due to NUMB or NUMBL downregulation. These differences in gene regulation extend to pathways, such as WNT or Hedgehog. In addition to these differences, the downregulation of either gene triggers a cancer stem cell-like related phenotype. These results show the importance of both genes as an intersection with different effects over cancer stem cell signaling pathways. www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 10), pp: 9219-9234 INTRODUCTION NUMB and NUMBL, or NUMB-Like, belongs to a closely conserved family of proteins with important roles in a large variety of cellular processes ranging from cell adhesion to ubiquitination [1–7]. NUMB, the first gene in the family identified in Drosophila [8], has been suggested to play a role in asymmetric division, allowing cell differentiation [7]. Most research conducted to date has been focused on the role of NUMB, assuming that NUMBL performs the same functions, although NUMBL does not display an asymmetric distribution in cells during mitosis [9]. In addition, its expression is different during development, with ubiquitous NUMB expression and more restricted expression of NUMBL in the central nervous system [6, 9–11]. Knock-out experiments in mice have shown that, although NUMBL deletion showed no differences during embryogenesis, the deletion of NUMB or combined NUMB/NUMBL deletion were embryonic lethal [6, 12, 13]. Together, these differences show that, although NUMB and NUMBL have a conserved structure and domains [14], the functional differences between the proteins must also be considered. NUMB and NUMBL have been characterized as tumor suppressor genes [15–17], leading to Notch signaling pathway inhibition [4, 17] or p53 stabilization [18, 19]. NUMB inhibits the Notch pathway through its interaction with ITCH and NICD (Notch IntraCellular Domain), labeling NICD for ubiquitination and degradation [4, 20–22]. Although this is one of the most known roles of NUMB, this protein has also been linked to the WNT pathway, promoting β-catenin degradation through polyubiquitination [23]. The role of NUMB as a tumor suppressor gene has been widely characterized, revealing that lower NUMB levels are associated with a worse prognosis in malignant pleural mesothelioma [24]. In addition, different tumors, such as breast cancer, salivary gland carcinoma, non-small-cell lung carcinoma or medulloblastoma, also exhibit a downregulation of NUMB expression [25–28]. Conversely, NUMB Research Paper Oncotarget9220 www.impactjournals.com/oncotarget overexpression reduces cell proliferation and increases cell sensitivity to cisplatin [24, 25, 29]. Previous results obtained by NUMBL knockdown by shRNA, with no changes in NUMB levels, showed an increment in tumorigenic properties and increased resistance to chemotherapy, with a worse prognosis in breast, lung and colorectal tumors [17]. Importantly, the downregulation of NUMBL also triggers Notch pathway activation, further increasing the epithelia-mesenchymal transition (EMT), cancer stem cell (CSC) transcriptional markers and CSC- like phenotypes. NUMBL has also been described as a tumor suppressor gene, mainly based on its ability to inhibit the Notch pathway [17, 30, 31]. However, NUMBL can also activate Hedgehog signaling, which represents a functional difference compared with NUMB [32]. According to these results, NUMBL can activate Hedgehog signaling and thus increase the stem cell population. This phenomenon suggests that, under certain circumstances, NUMBL could act as an oncogene. This process has also recently been described for NUMB, showing that an altered isoform expression is common in cancer cells [23, 33–35]. A small percentage of human tumors exhibit lower NUMBL expression than normal tissue, being this reduced expression associated with a poor prognosis and worse patient survival [30, 31]. Inhibition of only one NUMB family protein is sufficient to modify cancer cell properties, since a partial decrease in NUMB or NUMBL is sufficient to increase Notch pathway activation and cancer stem-like properties. This phenomenon suggests that NUMB and NUMBL act as essential regulators of cancer cell properties, individually acting in a dosedependent manner and regulating the same pathway with a certain degree of redundancy. Like NUMB, NUMBL seems to regulate Notch pathway activity [36, 37]. It is interesting to note that the downregulation of only one of these proteins, either NUMB or NUMBL, is sufficient to allow Notch pathway activation, increasing the pool of CSC-like cells [38–41]. However, whether there is a dose effect of combined NUMB/NUMBL or whether the downregulation of both proteins may coexist in some very aggressive tumors remains unknown. Such an additive effect still must be demonstrated, as well as whether the accumulative effects are linear or reach a certain threshold with no further increases in tumorigenicity. Finally, the effects of the distinct tumor suppressor activities on different pathways in cells must be elucidated. Here, we showed the mutual exclusivity of NUMB and NUMBL in tumors and the opposite gene regulation affecting at least three different signaling pathways: Notch, WNT and Hedgehog. Although both genes equally regulate Notch, the WNT and Hedgehog pathways are oppositely regulated by NUMB and NUMBL, according to our gene transcription analysis. However, the final phenotypic endpoint induced by NUMB or NUMBL downregulation was similar, suggesting some hierarchy in the signaling pathways. RESULTS NUMB and NUMBL show a negative correlation in tumors As mentioned above, NUMB and NUMBL are commonly described as proteins with a very similar function, characterized as tumor suppressors. To identify any differential effects in tumors, we analyzed a total of 95 datasets in ten different tumors (Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, ovarian colon/rectal, lung, renal breast, neuroblastoma or esophageal tumors) using R2 software. We observed a clearly marked trend between NUMB and NUMBL since were negatively correlated in 72 of the 95 considered datasets (Figure 1A). Indeed, the correlation of NUMB and NUMBL expression provided a significant Pearson value (R = –0.355, p = 4.4 × 10 –4 ) (Figure 1B), as clearly shown in the heat maps of tumors from colon, lung, breast, endometrium or kidney, in which individual samples can be compared (Figure 1C). These results, which were obtained directly from human tumor samples, showed that when one of these genes was highly expressed in tumors, the other was expressed at a lower rate. In fact, we also explored datasets derived from normal samples. We found that the negative correlation of NUMB/NUMBL was also present in non-tumoral samples, showing that both genes are to some extent mutually exclusive in cells (Supplementary Figure 1). This result suggests a stringent gene regulation to avoid undesirable effects. These differences might be due to differences in gene methylation, allowing higher expression of one of the genes in cells. To explore this phenomenon, we looked for changes in NUMB or NUMBL methylation using TCGA Wanderer resource [42]. By doing this, we found that NUMB is highly methylated in tumors regarding control (Figure 2A). However, the opposite happens with NUMBL, being highly methylated in normal samples but not in tumoral samples (Figure 2B). Differences in NUMB or NUMBL methylation between normal and tumoral samples appear to be general, as can be deduced from the fact that a common patron is obtained using four different CG probes (Figure 2C). At that way, for NUMB, methylation is higher in tumoral samples regarding control samples, while for NUMBL occurs the opposite. In addition, if we focus in the probe cg01582648, the percentage of tumoral samples with a methylation beta value higher than 0.8 is significantly higher regarding normal samples in breast invasive carcinoma (80.9% vs. 33.3%), colon adenocarcinoma (69.8% vs. 5.2%) and lung adenocarcinoma (88.8% vs. 71.6%) (Figure 2D). Again, the opposite happen for NUMBL probe cg20525355, being tumoral samples lower methylated regarding normal samples in colon adenocarcinoma (61% vs. 94.7%) and lung adenocarcinoma (75.8% vs. 100%) (Figure 2E). These results point to the possibility that NUMB or NUMBL Oncotarget9221 www.impactjournals.com/oncotarget might be preferentially expressed in a specific cell or tissue. Furthermore, suggest certain redundancy in the roles. NUMB and NUMBL have few common genes with the same correlations To identify the genes that correlated to NUMB and/or NUMBL in the same tumors, we performed the following analysis. We first selected public datasets from transcriptomic analyses of lung, cervical, breast and colorectal tumors (Supplementary Table 1). Next, we determined the genes that correlated to NUMB or NUMBL in all considered tumor types and then compared the individual tumor types to obtain a map of common genes correlating to NUMB or NUMBL expression in various tumors. We found 675 genes that were positively correlated and 691 genes that were negatively correlated to NUMBL in at least three of the considered tumor types. Regarding NUMB, we found 350 genes that were positively correlated and 108 genes that were negatively correlated, taking into account at least three of the tumors (Figure 3, Supplementary Table 2). Due to the previously assumed overlapping functions of NUMB and NUMBL in cells, it would be expected to find genes that were positively or negatively correlated to NUMB and NUMBL. In our bioinformatics analysis of tumors considering all tumor types, no common genes from public transcriptome tumor datasets were positively correlated both to NUMB and NUMBL. However, we obtained a short list of 5 genes that were negatively correlated both to NUMB and NUMBL (as expected from its behavior as tumor suppressors), a second list of 21 genes that were positively correlated to NUMB and negatively correlated to NUMBL, and two genes negatively correlated to NUMB and positively correlated Figure 1: NUMB and NUMBL are negatively correlated in human tumors. (A) NUMB/NUMBL correlations in leukemia, ovarian, colon, lung, kidney, breast, neuroblastoma and esophagus tumors, showing a higher percentage of negative correlations. (B) Correlation of NUMB and NUMBL expression in human tumors, showing a significant Pearson correlation coefficient (R = –0.355, p = 4.4 × 10–4). (C) Heat map of NUMB and NUMBL expression in human tumors. Oncotarget9222 www.impactjournals.com/oncotarget to NUMBL (Figure 4). However, when we compared the genes that were correlated to NUMB or NUMBL in each individual tumor type, we found common positively or negatively correlated genes, suggesting that NUMB and NUMBL participate in different gene regulatory processes, depending on the tissue (Supplementary Table 3). Gene ontology analysis of NUMB and/or NUMBL correlations Next, we performed Gene Ontology (GO)-term and Reactome enrichment analysis, considering separately the genes correlated to NUMB or NUMBL, due to the low Figure 2: Changes in NUMB/NUMBL methylation in tumoral samples. (A) Percentage of NUMB methylation for cg0158648 probe in six different tumors. Breast: Breast invasive carcinoma; COAD: Colon adenocarcinoma; READ: Rectum adenocarcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; Cervix: cervical squamous cell carcinoma and endocervical adenocarcinoma. (B) Percentage of NUMBL methylation for cg20525355 probe in six different tumors. (C) NUMB and NUMBL are differentially methylated in normal or tumoral samples. Red color: tumoral samples with a lower methylation value regarding normal samples. Blue color: tumoral samples with a higher methylation value regarding normal samples. (D) Relation between expression and methylation value for NUMB, using cg0158648 probe, in breast invasive carcinoma, colon adenocarcinoma and lung adenocarcinoma. (E) Relation between expression and methylation value for NUMBL, using cg20525355 probe, in breast invasive carcinoma, colon adenocarcinoma and lung adenocarcinoma. The Student’s T test was used to determine significant differences (*= p < 0.05; **= p < 0.01; ***= p < 0.001). Oncotarget9223 www.impactjournals.com/oncotarget number of common genes (Supplementary Table 4–7). Interestingly, we obtained common GO and Reactome terms for genes negatively correlated with NUMB or NUMBL (Supplementary Table 8). In particular, we found negative correlations with protein synthesis (from ribosome to mRNA maturation), general metabolism or protein transport. In addition, we found Reactome terms related to cell cycle, suggesting a possible deregulation due to changes in NUMB or NUMBL expression (Supplementary Table 8). At that way, one of the common genes negatively correlated both to NUMB and NUMBL was CDK4, a known gene connected to cell division. In addition, we also found components (MCM3 for NUMBL and MCM2 for NUMB) of the minichromosome maintenance protein (MCM) complex, necessary for cell cycle transition to S-phase [43]. These results can be related to the functions of NUMB and NUMBL as negative regulators of cell division, supporting their roles as tumor suppressor genes. From the list of genes that positively correlated with either NUMB or NUMBL, similar GO-terms could not be obtained. However, it is interesting to note that, regarding NUMB, some GO-terms were related to vesicle transport, a previously known NUMB function [44, 45] (Table 1). However, NUMBL appeared to be positively correlated to genes related to gene regulation and RNA metabolism (Table 2), suggesting an unexplored role for this gene. Genes that were negatively correlated to NUMBL resulted in a very extensive list of GO and Reactome terms, some of which were related to three different signaling pathways: NF-κB, WNT and Hedgehog, suggesting that, as previously reported [32], NUMBL could modulate the Hedgehog pathway (Table 3). We also found that NUMBL was negatively correlated to NF-kB signaling, Figure 3: Venn diagram of genes positively and negatively correlated to NUMB or NUMBL in breast, lung, colon and cervix tumors (p-value < 0.05). Oncotarget9224 www.impactjournals.com/oncotarget mitochondrial tricarboxylic acid cycle (TCA) and p53 stabilization, as also previously described [30, 46, 47]. NUMB and NUMBL show opposite correlations in some signaling pathways NUMB and NUMBL have been previously described as inhibitors of the Notch pathway due to their interactions with NICD, allowing it to be labeled for ubiquitination and degradation. However, we did not find any GO or Reactome term clearly related to the Notch pathway, so we decided to search specifically for genes related to this pathway. We also searched for genes in the WNT and Hedgehog pathways since both pathways were negatively correlated with NUMBL (Table 3). A significant percentage of genes related to the Notch pathway showed the same behavior according to the presence of NUMB or NUMBL (Figure 5). Genes such as CFLAR, EP300, GBP2, HEYL, KLF7, NOTCH1 or POFUT showed a similar correlation with NUMB or NUMBL in the three tumor types considered (Figure 5). However, many of the genes correlating with NUMBL showed an opposite behavior with NUMB in the other two analyzed pathways, WNT and Hedgehog (Figure 5), independently of the type of tumor. These results indicated that NUMB and NUMBL could differentially modulate different signaling pathways in a very different fashion. These results also indicate that besides the previously observed exclusivity, the full redundancy does not exist between these two genes. Only in the Notch pathway, equal regulation is maintained. To experimentally assess this opposite gene regulatory role, we decided to analyze gene expression using T47D and HeLa cells transfected with specific shRNAs against NUMB or NUMBL (Figure 6A). Focusing on genes in the Notch pathway, we observed an equivalent activation of this pathway due to the downregulation of NUMB or NUMBL (Figure 6B), correlating with previous reports [17, 48]. Due to the relevance of the different gene associations with NUMB and NUMBL, we extended our analysis to the other two signaling pathways, WNT and Hedgehog. As expected from the bioinformatics analysis, we found opposite results due to the downregulation of NUMB or NUMBL in the mRNA levels of the target genes TCF4, EN1, SOX9 and BTRC, which are considered readouts of the WNT pathway (Figure 6C); or PTCH1, GLI1, GLI2 and CDH1, considered read-outs of the Hedgehog pathway (Figure 6D). Our bioinformatics results also led us to analyze other genes with opposite correlations in tumors, such as NFκB1, ERBB2 and TEAD1. Measurement of the transcriptional responses of these genes to the reduced expression of NUMB or NUMBL revealed an opposite regulation of these genes in response to NUMB or NUMBL (Figure 6E), as expected based on our bioinformatics analysis. It is Figure 4: Venn diagram of genes positively or negatively correlated to NUMB or NUMBL, showing coincident genes. Oncotarget9225 www.impactjournals.com/oncotarget interesting to note that these three genes are part of other signaling pathways (NFκB, ERBB/HER and Hippo) [49– 51], likely suggesting complex interaction of NUMB/L proteins with the signaling network. NUMB and NUMBL downregulation equally increases stem cell properties Although NUMB and NUMBL downregulation act differently regarding some of the considered genes, suggesting that NUMB and NUMBL target some different pathways, the global effect caused by their downregulation is very similar. To explore this point, we decided to measure the expression of some genes associated with the acquisition of stem cell-like properties and observed a clear increment in SOX2, BMI1 and NANOG genes in response to both NUMB and NUMBL downregulation (Figure 7A), which have been previously related to stem cell properties [52–54]. The increment in the transcription of stem celllike genes, together with the previously described increment in other stem cell-like genes related to NUMB or NUMBL downregulation, such as OCT4 or KLF4, showed that, for all analyzed conditions, the cells acquire a more dedifferentiated state, typical of stem-like cells [17, 48]. To rate these stem cell-like properties, we cultured cells at a low density, so that individual clones formed independent colonies. These clones can be classified as holoclones, meroclones and paraclones, according to their ability to reconstitute a tumor [55, 56]. Holoclones are considered to derive from cells with stem cell-like properties. We found that either NUMB or NUMBL downregulation caused similar increments in the Table 1: GO-terms obtained from genes positively correlated with NUMB GO-term Fold enrichment p-value vesicle targeting 8.47 2.76 × 10–4 COPII vesicle coating 8.37 0.0161 vesicle targeting, rough ER to cis-Golgi 8.37 0.0161 vesicle coating 8.12 0.0207 COPII-coated vesicle budding 7.87 0.0264 vesicle targeting, to, from or within Golgi 7.76 0.0298 actomyosin 6.9 0.0372 establishment of vesicle localization 4.76 3.19 × 10–4 vesicle localization 4.53 6.87 × 10–4 Golgi vesicle transport 4.04 2.34 × 10–4 Vesicle organization 3.47 0.0341 vesicle-mediated transport 2.27 5.75 × 10–3 Golgi subcompartment 2.27 0.0332 Golgi apparatus 2.08 5.66 × 10–4 cytoplasmic vesicle 1.85 4.58 × 10–4 intracellular vesicle 1.85 4.82 × 10–4 vesicle 1.53 1.58 × 10–3 Table 2: GO-terms obtained from genes positively correlated with NUMBL GO-term Fold Enrichment p-value transcription, DNA-templated 1.89 1.31 × 10–8 nucleic acid-templated transcription 1.89 1.35 × 10–8 RNA biosynthetic process 1.88 1.71 × 10–8 nucleobase-containing compound biosynthetic process 1.72 1.94 × 10–6 DNA binding 1.71 4.75 × 10–5 RNA metabolic process 1.64 3.86 × 10–6 regulation of RNA metabolic process 1.53 1.30 × 10–4 regulation of RNA biosynthetic process 1.51 1.25 × 10–3 regulation of transcription, DNA-templated 1.51 1.25 × 10–3 Oncotarget9226 www.impactjournals.com/oncotarget percentages of holoclones of HeLa and T47D cells (Figure 7B). In addition, tumorspheres, colonies derived from stem cell-like cells that grow in suspension, showed a significant increment in number in both cell lines, demonstrating again that NUMB/NUMBL downregulation increased stem cell-like properties (Figure 7C), regardless of the differences in pathway activation. DISCUSSION NUMB and NUMBL have been considered tumor suppressor genes with very similar functions because both can regulate the Notch pathway [5, 17, 22, 30, 57–59]. Our initial analysis in human tumors showed that both genes are negatively correlated in more than 75% percent of the considered datasets (72 out 95 different datasets). In addition, their expression in non-tumoral samples, which is close to 75%, shows that this opposite regulation is a common effect in human cells. Their role as negative regulators of the Notch pathway likely requires a higher transcription of one of the genes than the other, allowing only one of the two proteins to effectively perform its regulatory role. This notion is supported by development studies conducted in mouse and chicken embryos, where NUMB is broadly expressed, while NUMBL appears to be enriched in the developing central nervous system [9–11]. Our previous results have focused on the downregulation of NUMB or NUMBL by shRNA [17, 48]. We showed that only that the downregulation of only NUMB or NUMBL causes an increment in the tumorigenic properties of cells, allowing us to focus on their roles as tumor suppressor genes. Therefore, cells with lower NUMBL levels are more resistant to drugs that are commonly used in cancer chemotherapy, such as doxorubicin or vincristine [17]. In fact, loss of NUMBL protein has been associated with an increase in metastasis and worse survival [60]. Our bioinformatics analysis allowed us to obtain an unexpected network of gene correlations with NUMB or NUMBL. Due to their accepted roles as genes with similar functions, we expected to find common correlations for both genes in different tumors. However, our analysis showed that NUMB and NUMBL exhibited Table 3: GO and Reactome pathways terms related to Hedgehog, WNT and NF-κB pathways, obtained from genes negatively correlated with NUMBL GO-term Fold enrichment p-value NIK/NF-kappaB signaling 5.15 3.62 × 10–3 respiratory electron transport chain 4.55 4.37 × 10–5 Wnt signaling pathway, planar cell polarity pathway 4.68 5.27 × 10–3 Reactome pathways term Entities p-Value Entities FDR Hedgehog ‘off’ state 1.72 × 10–3 0.0137 GLI3 is processed to GLI3R by the proteasome 3.06 × 10–6 9.18 × 10–5 Degradation of GLI2 by the proteasome 3.06 × 10–6 9.18 × 10–5 Degradation of GLI1 by the proteasome 3.06 × 10–6 9.18 × 10–5 Hedgehog ligand biogenesis 1.74 × 10–5 2.44 × 10–4 Hh mutants that don’t undergo autocatalytic processing are degraded by ERAD 1.16 × 10–5 2.06 × 10–4 Hh mutants abrogate ligand secretion 2 × 10–5 2.65 × 10–4 Beta-catenin independent WNT signaling 2.63 × 10–3 0.021 Degradation of beta-catenin by the destruction complex 1.7 × 10–5 2.44 × 10–4 Degradation of AXIN 5.47 × 10–6 1.26 × 10–4 Autodegradation of Cdh1 by Cdh1:APC/C 3.7 × 10–6 9.62 × 10–5 Dectin-1 mediated noncanonical NF-kB signaling 2.72 × 10–5 3.26 × 10–4 Activation of NF-kappaB in B cells 6.81 × 10–5 6.81 × 10–4 TNFR2 non-canonical NF-kB pathway 2.4 × 10–3 0.0192 NIK––>noncanonical NF-kB signaling 1.16 × 10–5 2.06 × 10–4 Citric acid cycle (TCA cycle) 2.58 × 10–4 2.58 × 10–3 The citric acid (TCA) cycle and respiratory electron transport 5.75 × 10–9 9.03 × 10–7 Stabilization of p53 1.69 × 10–6 7.61 × 10–5 p53-Dependent G1 DNA Damage Response 1.26 × 10–5 2.06 × 10–4 Oncotarget9227 www.impactjournals.com/oncotarget almost independent correlations when we considered the four tumor types. This result could be due to different regulatory mechanisms, depending on the tissue in which NUMB or NUMBL is expressed. A comparison of each tumor type allowed us to identify a small number of genes that were equally related to NUMB and NUMBL, different genes related to similar functions, and genes with differential expression. GO-terms obtained with the different lists of genes that were correlated to NUMB described functions such as endocytic protein with important roles in protein transport [2, 3, 44, 45]. GO- terms related to NUMBL were related to gene transcription and some of its previously described roles [17, 31, 61]. In addition, common GO and Reactome terms obtained from negative correlations to NUMB or NUMBL proceeded from different list of genes, pointing to a different gene regulation network. These data may confirm the different cellular roles of each protein. The Notch pathway is involved in the maintenance of tumor stemness and cancer metastasis. This pathway is activated in different tumors, such as lung, colon, breast and prostate tumors, and in sarcomas, melanomas, leukemias and lymphomas [58, 59, 62–65]. In addition, Notch activity has been linked to cancer metastasis by inducing EMT, tumor angiogenesis processes and anoikis resistance of tumor cells [66, 67]. After its interaction with a ligand, Notch is proteolytically cleaved, and NICD is released into the cytoplasm, allowing its nuclear translocation [68, 69]. We have recently showed that NUMBL behaves, such as NUMB, its close relative, as a tumor suppressor gene regulating the Notch pathway. Here, we showed that NUMB and NUMBL presented mutual exclusivity in tumors, suggesting that the tumor suppressor effect on the Notch pathway is regulated by one protein at a time, thus simplifying the regulatory network. In addition, NUMB and NUMBL also showed opposite correlations in non-tumoral samples, suggesting that this opposite relationship is a common effect in cells. However, the two proteins, which share approximately 55% of their amino acid sequence, did not show total redundancy, as deduced from the differences in mRNA levels observed for genes belonging to the NFκB, ERBB/ HER, Hippo, WNT and Hedgehog pathways. Thus, our results obtained by downregulation of NUMBL are consistent with previously published studies showing that NUMBL negatively influences the NFκB pathway and positively affects the Hedgehog pathway [31, 32]. In addition, although GLI1 and GLI2 should have similar behaviors, considering their transcription is activated by Hedgehog signaling and appear to be downregulated when the Hedgehog pathway is inactive, GLI1 is also connected to Notch signaling through HES1 inhibition [70, 71]. The large number of genes that are transcriptionally affected in an opposite manner suggests that NUMB and NUMBL Figure 5: Upper figure: Notch, Hedgehog and WNT pathway genes with an opposite correlation for NUMB and NUMBL. Statistical differences, obtained from gene correlations, are the followings: *= p < 0.05; **= p < 0.01; ***= p < 0.001. Bottom figure: Graph representative of the genes equally regulated vs. opposite regulated for each pathway in breast, lung and colon tumors. Oncotarget9234 www.impactjournals.com/oncotarget 61. Vaira V, Faversani A, Martin NM, Garlick DS, Ferrero S, Nosotti M, Kissil JL, Bosari S, Altieri DC. Regulation of Lung Cancer Metastasis by Klf4-Numb-like Signaling. Cancer research. 2013; 73:2695–2705. 62. Leong KG, Gao WQ. The Notch pathway in prostate development and cancer. Differentiation. 2008; 76:699–716. 63. Pierfelice TJ, Schreck KC, Eberhart CG, Gaiano N. Notch, Neural Stem Cells, and Brain Tumors. Cold Spring Harbor symposia on quantitative biology. 2008; 73:367–375. 64. Wang Z, Li Y, Banerjee S, Sarkar FH. Emerging Role of Notch in Stem Cells and Cancer. Cancer Letters. 2009; 279:8–12. 65. Zweidler-McKay PA. Notch signaling in pediatric malignancies. Current Oncology Reports. 2008; 10:459–468. 66. Hu YY, Zheng MH, Zhang R, Liang YM, Han H. Notch Signaling Pathway and Cancer Metastasis. In: Reichrath J, Reichrath S, eds. Notch Signaling in Embryology and Cancer. (New York, NY: Springer US), pp. 186–198. 67. Yumei L, Jia M, Xiujuan Q, Qiong W, Jun X, Lucio M, Fazlul HS, Zhiwei W. Regulation of EMT by Notch Signaling Pathway in Tumor Progression. Current Cancer Drug Targets. 2013; 13:957–962. 68. Mumm JS, Schroeter EH, Saxena MT, Griesemer A, Tian X, Pan DJ, Ray WJ, Kopan R. A Ligand-Induced Extracellular Cleavage Regulates gamma-Secretase-like Proteolytic Activation of Notch1. Molecular Cell. 2000; 5:197–206. 69. Bertrand FE, McCubrey JA, Angus CW, Nutter JM, Sigounas G. NOTCH and PTEN in prostate cancer. Advances in Biological Regulation. 2014; 56:51–65. 70. Mazumdar T, DeVecchio J, Agyeman A, Shi T, Houghton JA. The GLI genes as the molecular switch in disrupting Hedgehog signaling in colon cancer. Oncotarget. 2011; 2:638–645. https://doi.org/10.18632/oncotarget.310. 71. Schreck KC, Taylor P, Marchionni L, Gopalakrishnan V, Bar EE, Gaiano N, Eberhart CG. The Notch Target Hes1 Directly Modulates Gli1 Expression and Hedgehog Signaling: A Potential Mechanism of Therapeutic Resistance. Clinical cancer research. 2010; 16:6060–6070. 72. Schroeder MP, Gonzalez-Perez A, Lopez-Bigas N. Visualizing multidimensional cancer genomics data. Genome Med. 2013; 5:9. 73. Marcotullio LD, Ferretti E, Greco A, De Smaele E, Po A, Sico MA, Alimandi M, Giannini G, Maroder M, Screpanti I, Gulino A. Numb is a suppressor of Hedgehog signalling and targets Gli1 for Itch-dependent ubiquitination. Nat Cell Biol. 2006; 8:1415–1423. 74. Beres BJ, George R, Lougher EJ, Barton M, Verrelli BC, McGlade CJ, Rawls JA, Wilson-Rawls J. Numb regulates Notch1, but not Notch3, during myogenesis. Mechanisms of Development. 2011; 128:247–257. 75. Oliveros JC. Venny. An interactive tool for comparing lists with Venn’s diagrams. http://bioinfogp.cnb.csic.es/tools/ venny/index.html (2007–2015).