Numb-like (NumbL) downregulation increases tumorigenicity, cancer stem cell-like properties and resistance to chemotherapy

Oncotarget63611 www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 39 Numb-like (NumbL) downregulation increases tumorigenicity, cancer stem cell-like properties and resistance to chemotherapy José M. García-Heredia1,2, Eva M. Verdugo Sivianes1, Antonio Lucena-Cacace1, Sonia Molina-Pinelo1,3, Amancio Carnero1 1Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocio, Universidad de Sevilla, Consejo Superior de Investigaciones Cientificas, Seville, Spain 2Department of Vegetal Biochemistry and Molecular Biology, University of Seville, Seville, Spain 3Present address: Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain Correspondence to: Amancio Carnero, email: [email protected] Keywords: NumbL, Notch, cancer stem cells, tumor suppressor, tumorigenicity Received: April 22, 2016 Accepted: August 12, 2016 Published: August 23, 2016 ABSTRACT NumbL, or Numb-like, is a close homologue of Numb, and is part of an evolutionary conserved protein family implicated in some important cellular processes. Numb is a protein involved in cell development, in cell adhesion and migration, in asymmetric cell division, and in targeting proteins for endocytosis and ubiquitination. NumbL exhibits some overlapping functions with Numb, but its role in tumorigenesis is not fully known. Here we showed that the downregulation of NumbL alone is sufficient to increase NICD nuclear translocation and induce Notch pathway activation. Furthermore, NumbL downregulation increases epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-related gene transcripts and CSC-like phenotypes, including an increase in the CSC-like pool. These data suggest that NumbL can act independently as a tumor suppressor gene. Furthermore, an absence of NumbL induces chemoresistance in tumor cells. An analysis of human tumors indicates that NumbL is downregulated in a variable percentage of human tumors, with lower levels of this gene correlated with worse prognosis in colon, breast and lung tumors. Therefore, NumbL can act as an independent tumor suppressor inhibiting the Notch pathway and regulating the cancer stem cell pool. INTRODUCTION NumbL, or Numb-like, is a close homologue of Numb and is part of an evolutionary conserved protein family implicated in some important cellular processes, including cell adhesion and migration, asymmetric cell division, and targeting proteins for endocytosis and ubiquitination [1–7]. Numb was originally identified as a membrane-associated protein (dNumb) in Drosophila mutants with severe defects in neuron development [8]. This protein is asymmetrically segregated during telophase to only one daughter cell, acting as a cell fate determinant. The daughter cell with dNumb will differentiate while the cell without dNumb will remain in an undifferentiated state [6]. dNumb is evolutionarily conserved, with its mammalian homologues encoded by two genes, Numb and NumbL [9]. Although vertebrate Numb also shows asymmetric distribution in cells, NumbL has been shown to be symmetrically distributed in cytoplasm, which differentiates it from its close homologue Numb [10]. In addition, Numb is ubiquitously expressed during development, while NumbL is more restricted to the developing central nervous system [7, 10–12]. These and other studies suggest that Numb and NumbL, despite their similarities, maintain at least some independent functions. This is more obvious during development in mice. NumbL removal does not have any apparent effects in mice lacking the gene, with the exception of a reduction in female fertility [7]. However, Numb knock-out or double Numb/NumbL knockouts lead to demise in mice during development; the phenotype with double knockout is more severe in terms of early embryonic lethality, showing that Numb/NumbL partially overlap but have some important differences [7, 13, 14]. Accumulating evidence suggests a potential role of Numb as a tumor suppressor [15, 16], including inhibition of the Notch signaling pathway [17] and the stabilization of p53 [18, 19]. Loss of Numb Research Paper Oncotarget63612 www.impactjournals.com/oncotarget was associated with poor prognosis in malignant pleural mesothelioma [20]. Overexpression of Numb significantly inhibits proliferation, enhances apoptosis and increases sensitivity to cisplatin [20]. In Numbnegative breast and clear cell renal carcinoma cells, ectopic overexpression of Numb suppresses proliferation [21, 22]. In vivo, RNA interference of Numb in a model of mouse lymphomagenesis accelerates the onset of lymphomas [23]. Loss of Numb expression has also been reported in some types of human cancer, including breast, NSCLC, and salivary gland carcinomas and medulloblastoma [21, 24–26]. However, there is a trend toward higher Numb expression in more malignant tumors in human astrocytomas [27]. Overexpression of Numb has also been observed in cervical squamous carcinoma cells [28], suggesting that Numb may act as an oncogene in certain tissues. In mammals, six differentially spliced isoforms of Numb, each with different expression patterns, have been described to date, while only one NumbL protein has been identified [29, 30]. This suggests that certain Numb isoforms (isoforms 2, 4, 5 and 6) promote tumor growth [31–33], while the Numb-1 isoform behaves as a tumor suppressor [34] and Numb-3 behaves as an oncogene or tumor suppressor depending on the tissue [35]. This raises the possibility that the role of Numb is isoform-specific. Considerably less is known about mammalian NumbL and its role in tumorigenesis. NumbL was reported to be downregulated in lung cancer cell lines; its ectopic expression suppresses proliferation and invasion, increasing apoptosis [36]. Similarly, in human glioblastoma cells, the overexpression of NumbL suppressed, while the elimination of NumbL promoted the migration and invasion of glioma cells [37]. However, another report found that increased deregulated expression of NumbL in lung tumor cell lines mediated cell migration and in human tumors correlates with shorter patient survival [38]. Therefore, there are contradictory data about the role of NumbL in cancer progression. Our aim in this study was to investigate the role of NumbL in tumorigenesis, specifically the control of the CSC phenotype, which has emerged as a preferred target in cancer therapy because of its role in cancer recurrence. Here we show that NumbL knockdown increases tumorigenic properties in three different cancer cell lines of different origins, cervix HeLa, breast T47D and sarcoma AX, due to Notch pathway activation by stabilizing NICD. NumbL knockdown is sufficient to induce cancer stem cell-like transcription and phenotypic properties, suggesting an important role as a tumor suppressor gene by maintaining the CSC-like phenotype through Notch pathway activation. Furthermore, low levels of NumbL decrease sensitivity to chemotherapy and correlate to a worse prognosis in breast, lung and colorectal tumors. RESULTS shRNA against NumbL induces protein downregulation and a higher number of colonies in clonogenic assays Human cell lines HeLa and T47D transfected with shRNA against NumbL (shNbL2) showed a decrease in the expression of this protein, both at mRNA and protein levels (Figure 1A). Despite the high degree of similarity between Numb and NumbL, cell lines expressing shNbL2 show no significant changes in Numb expression (Figure 1B). Clonogenic assays performed at low cell densities showed that NumbL downregulation induces a significant increment in colony numbers, both from HeLa and T47D cells (Figure 1C). Colony numbers from HeLa cells were increased to 140%, while for T47D cells, NumbL downregulation induced a further significant increase up to values close to 230% compared to the control expressing a scrambled shRNA cell line. This increment in colony number has been associated with increased cell survival. Further experiments growing these cells in soft agar also showed a clear increase in the number of colonies growing in cells with downregulated NumbL (Figure 1D). To further confirm the role of NumbL as a tumor suppressor, we overexpressed NumbL cDNA into these cells. The experiment showed a marked reduction in the number of cells able to form colonies (Figure 1E). These data confirm the potential of NumbL to act independently as a tumor suppressor. NumbL downregulation activates the Notch pathway Due to its close relationship to Numb and the known connection between Numb and Notch, we decided to analyze the effect of NumbL downregulation on the Notch pathway. We detected an increment in nuclear NICD in HeLa and T47D cells with low levels of NumbL compared to the control cells (Figure 2A). This increase is similar to the increase observed upon Numb downregulation by shRNA (Figure 2A). As a consequence of NICD nuclear translocation, the Notch pathway is activated. We found Hes1/Hes5 activation and Gli1 inhibition, as well as Klf7 and Id2 inhibition (Figure 2B), all wellknown downstream genes regulated by NICD and Notch pathways [39–42]. Taken together, these data suggest that NumbL downregulation induces Notch pathway activation by protecting NICD from degradation. Acquisition of stem cell-like properties due to NumbL downregulation It has been previously established that activation of the Notch pathway induces EMT and cancer stem celllike properties [43]. Using RT-qPCR, we analyzed the expression of some genes that have been connected with Oncotarget63613 www.impactjournals.com/oncotarget the acquisition of EMT and stem cell-like properties. NumbL downregulation induced a clear increment in EMT-dependent transcript Snai1 and Twist1 in Hela and T47D cells (Supplementary Figure S1A). In both HeLa and T47D cells, we also observed a significant induction of Klf4, Sox2, Nanog, Oct4 and Bmi1 genes (Figure 3A). These genes have been previously related to stem cell properties [44–46]. The higher expression of these genes showed that downregulation of NumbL and, consequently, Notch pathway activation, turn cells into a certain de-differentiated state typical of stem-like cells. In addition, FACS analysis of HeLa and T47D cells with downregulated NumbL showed a higher expression of phenotypic markers associated with stem cells (Figure 3B and Supplementary Figure S2). For T47D cells, a breast tumor cell line, we used double CD44+/CD24− staining, which has been extensively used to identify cancer stem cells [47]. In this case, NumbL downregulation increased the percentage of CD44+/ CD24− from 13.6% of scrambled cells to 36.6% of cells expressing NumbL shRNA (Figure 3B). In addition, CD44 has been characterized as a target gene activated by the Notch signaling pathway [48]. In the case of HeLa cells, we used the CD133 stem cell marker to differentiate the CSC subpopulation [49]. CD133+ HeLa cells increased from 0.8% in control cells to 2.7% in cells expressing NumbL shRNA, indicating that NumbL downregulation in HeLa cells also induces the phenotypic markers of CSC- like cells. Analysis of cancer stem cell-like properties of cells with downregulated NumbL To this end, we cultured cells at low density to form independent colonies from individual clones. These clones have been previously classified as holoclones, meroclones and paraclones based on their ability to reconstitute a tumor from a single cell [50, 51]. Basically, holoclones are considered to be derived from stem cells, while paraclones are differentiated cells that are incapable of reconstituting a culture (Figure 4A). Meroclones are intermediate phenotypes between holo- and paraclones. The percentage of holoclones in shNbL cells was increased from 30% to 60% in HeLa cells, while in T47D this increment was from 30% to 50% (Figure 4A). Figure 1: (A) Transfection of HeLa or T47D cells with NumbL-shRNA plasmids induce a decrease in NumbL expression, detected both by RT-qPCR and WB assays. (B) This shRNA has no effect on Numb expression as determined by RT-qPCR assays. (C) Clonogenicity assays for HeLa and T47D pBABE cells transfected with scrambled or shNbL shRNA2, (D) soft agar, (E) growth clonability by overexpression of NumbL cDNA. All experiments were repeated a minimum of three independent times in triplicate. All figures include Student’s t-test for statistical analysis of the data. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Oncotarget63614 www.impactjournals.com/oncotarget Figure 2: NumbL downregulation activates Notch signaling. (A) NICD localization in nuclei and total extracts in HeLa and T47D cells. (B) Expression of Notch pathway-related genes. Two different shRNAs (shNbL2 and shNbL4) were used to eliminate possible off-target effects. The sequence of both shRNAs is shown in M&M. All experiments were repeated a minimum of three independent times in triplicate. All figures include Student’s T test for statistical analysis of the data. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Oncotarget63615 www.impactjournals.com/oncotarget Next, we tested the ability of cells with reduced levels of NumbL to form tumorspheres, another surrogated assay for the cancer stem-like phenotype. The cells were seeded and visualized five days after seeding. T47D tumor cells formed spheres at this stage, considered as 1st generation tumorspheres. There were morphological differences between tumorspheres from scrambled or shNbL cells carrying NumbL shRNA2 (Figure 4B). The number of tumorspheres derived from cells with reduced NumbL was significantly higher compared to the control scrambled shRNA. The analysis of the 2nd generation tumorspheres derived from HeLa cells showed even more evident differences between control and NumbL downregulated cells (Figure 4B). At this stage, only shNbL2 cells showed real tumorspheres (Figure 4B). In this case, the spheres were also larger when NumbL was downregulated. Finally, we decided to downregulate NumbL in a low passaged sarcoma cell line, AX. This cell line is close to the primary sarcoma tumor (liposarcoma) because it has been passaged less than 20 times from the original explant. This cell line behaves in a similar fashion to Hela and T47D regarding the increase of the stem cell-like pool upon NumbL downregulation (Figure 4). Overexpression of NumbL cDNA reduced Notch and stem cell gene transcription and stem cell-like properties To fully confirm the role of NumbL as a tumor suppressor acting on the Notch pathway and stem cell gene transcription, we decided to overexpress NumbL cDNA into these three cell lines and assess transcription and behavior. The overexpression of NumbL triggers a decrease in the stem cell genes and the inactivation of the Notch pathway (Figure 5A). As a consequence, NumbL overexpression decreases the number of holoclones (Figure 5B) and tumorspheres (Figure 5C), finally leading to a decrease in colony growth (Figure 1E). Figure 3: NumbL downregulation activates Notch stem-cell like properties in tumor cells. (A) Quantitative PCR results with genes related to stem-cell like properties. Two different shRNAs (shNbL2 and shNbL4) were used to eliminate possible off-target effects. Sequence of both shRNAs is shown in M&M. (B) CD44+/CD24− and CD133+ FACS of T47D and HeLa cells, respectively. All experiments were repeated a minimum of three independent times in triplicate. All figures include Student’s T test for statistical analysis of the data. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Oncotarget63616 www.impactjournals.com/oncotarget Figure 4: (A) Type and percentage of each of the different clones observed for HeLa, T47D and AX cells. Figure shows typical colonies (left) and the percentage of each type in all cases (right). (B) Tumorsphere assay for HeLa cells (2nd generation), T47D cells (1st generation), and AX low passaged sarcoma cells (1st generation) showing typical colonies (left) and both total number and size of tumorspheres (right). All experiments were repeated a minimum of three independent times in triplicate. All figures include Student’s T test for statistical analysis of the data. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Oncotarget63617 www.impactjournals.com/oncotarget Figure 5: Overexpression of NumbL cDNA in HeLa, T47D and AX cells. (A) Overexpression of NumbL cDNA downregulates stem- and Notch-pathway related genes. (B) Type and percentage of each of the different clones observed for HeLa, T47D and AX cells upon overexpression of NumbL cDNA. Figure shows typical colonies (left) and the percentage of each type in all cases (right). (C) Tumorsphere assay for HeLa cells (2nd generation), T47D cells (1st generation), and AX low passaged sarcoma cells (1st generation) showing total number of tumorspheres. All experiments were repeated a minimum of three independent times in triplicate. All figures include Student’s T test for statistical analysis of the data. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Oncotarget63618 www.impactjournals.com/oncotarget Finally, to explore the role of Notch activation, we separated the stem cell-like subpopulation from T47D, the CD44+/CD24− cells from the CD44- cells, and analyzed whether there was differential activation of the Notch pathway in these cells regarding the levels of NumbL. In the T47D control cells, we observed that stem cell gene transcription was increased in CD44+/CD24− cells; however, Notch-dependent transcription was inhibited, as denoted by the normal levels of Hes5 and the activation of Klf7 and Gli1 (Figure 6, upper panel). In the T47D with downregulated levels of NumbL, we found a marked activation of the Notch pathway in CD44+/CD24− cells, as denoted by the activation of Hes5 and the repression of Klf7 and Gli1 (Figure 6, bottom panel). We also observed differences in the levels of Snai1, suggesting that the EMT was dependent on Notch pathway activation. NumbL downregulation increases chemoresistance It has been proposed that the extent of chemoresistance is related to the percentage of CSCs in the tumor. Since downregulation of NumbL increases the CSC-like phenotype in culture, we tested whether this phenotype is also associated to chemoresistance. To this end, we treated Hela and T47D cells carrying NumbL shRNA (shNbL2 cells) or scramble (scr) to different doses of several chemotherapeutic drugs used in clinical regimens: doxorubicin, irinotecan, 5-FU and gemcitabine. After IC50 calculations, we observed that Hela cells with NumbL downregulated showed on average a two-fold increase in IC50 (Table 1), confirming the increase of resistance of these cells to the drug tested. In T47D, we did not observe an increase in IC50 throughout the different experiments performed (Table 1); however, we detected in most cases an increase in the percentage of cells resistant to the treatment (Figure 7). To explore this in more depth, we subjected T47D to new drugs most commonly used in breast tumors, including vincristine, capecitabine and paclitaxel. In all cases NumbL-downregulated T47D were more resistant to treatment (Table 1). This is especially relevant in the case of vincristine and paclitaxel, in which a large percentage of cells remained resistant to the treatment (Figure 7A). This resistance is not a general characteristic of the newly generated cell line with low NumbL because the sensitivity remained identical to control cells with other treatments such as sunitinib (Figure 7A). Because mammary tumor cells showed the Figure 6: T47D control cells or T47D cells expressing the shNbl2 were selected for CD44+/CD24− (solid black bars) or CD44- (open bars) subpopulations. Transcriptional analysis of stem- or Notch-related genes was performed by qRT-PCR. All experiments were repeated a minimum of three independent times in triplicate. All figures include Student’s T test for statistical analysis of the data. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Oncotarget63619 www.impactjournals.com/oncotarget highest ratio of resistance to vincristine treatment, we treated T47D cells with vincristine and subjected them to flow cytometry analysis to detect the population of CD44+/ CD24 − cells. The results indicated that resistant cells have an increased proportion of CD44+/CD24− cells being enriched in cancer stem-like cells, mainly when NumbL is downregulated (Figure 7B). NumbL downregulation in human tumors Our previous data strongly suggest that NumbL may behave as a tumor suppressor in human tissue. To explore this possibility, we searched public array databases (through Oncomine) for the possible downregulation of this gene expression in tumors. We found transcriptomic analysis showing that when comparing normal colon tissue to colorectal tumors, the tumors showed consistently lower expression of this gene (Supplementary Figure S3). Breast and lung tumor databases also exhibited a variable number of tumor samples with downregulated NumbL mRNA levels, which does not account for a significant decrease of the total tumor population (data not shown). This decrease in NumbL transcription may be a consequence of promoter methylation. To explore this point, we analyzed our own cohort of lung tumors (Supplementary Table S2). To evaluate the potential role of NumbL promoter methylation in lung cancer, we analyzed the methylation status of the cluster in human lung tissue. The methylation profile of NumbL was evaluated in human tumor samples and compared to non-tumor tissue using the Illumina Infinium Human Methylation 450 BeadChip. The methylation levels in lung cancer versus nontumor tissue are shown in Table 2. Our data show a significant methylation of the NumbL promoter in lung adenocarcinoma tumors compared to non-tumor lung samples. The same significant NumbL methylation profiles were observed in squamous cell lung cancer. Interestingly, the methylation of the NumbL promoter was more significant in smoker patient samples, while it was nonsignificant in samples from non-smoker patients. These data reinforce the role of NumbL as a tumor suppressor in human tumors, being downregulated in a variable subset of tumors depending on the tissue. Furthermore, in colon tumors, low levels of this gene are associated with worse prognosis (Figure 8A–8C and Supplementary Table S2). In breast and lung tumors, the small percentage of tumors with low NumbL mRNA levels also showed worse prognosis (Figure 8D–8H and Supplementary Table S2). However, the overall statistics were not significant values, likely due to the very different number of cases in both arms; yet, a clear tendency can be observed. A plausible explanation is that the tumors with downregulated NumbL have a larger number of cancer stem cells that avoid the action of the chemotherapy, thus providing an advantage for tumor relapse. When we specifically selected the relapsed tumors, those with low levels of NumbL had a worse prognosis, at least in breast tumors (Figure 9A and 9B); the relapses were both local and at distant sites (Figure 9C). DISCUSSION NumbL, a closely related homologue to Numb, has been recently linked to cancer. Our results strongly support a tumor suppressor role for NumbL. Downregulating NumbL without changing Numb allows cells to acquire a higher tumorigenic potential due to Notch pathway activation. Therefore, inhibition of only one of the Numb family proteins is sufficient to modify cancer cell properties. Importantly, we found that NumbL downregulation triggers the activation of the Notch pathway, further increasing the EMT and CSC transcriptional markers and CSC-like phenotypes. In human tumors, a number of tumors have a lower NumbL expression than normal tissue. This lower NumbL Table 1: IC50 (µM) values for Hela and T47D expressing shRNA scramble (scr) or shRNA against NumbL (shNbl) of different drugs commonly used in cancer chemotherapy HeLa T47D scr shNbL scr shNbL Doxorubicin 0.4 ± 1.12 0.8 ± 1.24 0.11 ± 1.09 0.12 ± 1.21 Irinotecan 27.6 ± 1.06 43.65 ± 1.09 2.21 ± 1.1 2.32 ± 1.34 5-FU 46 ± 1.11 94.4 ± 1.12 2.46 ± 2.31 3.54 ± 2.18 Gemcitabine 3.84 ± 1.46 10.23 ± 1.58 0.0091 ± 1.24 0.0067 ± 1.1 Vincristine 1.26 ± 0.35 2.71 ± 0.56 0.001 ± 0.001 Not reached Sunitinib NT NT 2.46 ± 2.31 3.54 ± 2.18 Capecitabine NT NT 466,96 ± 45.26 683,26 ± 70,36 Paclitaxel 0.125 ± 0.05 0.142 ± 0.03 0.0169 ± 0.001 0.9 ± 0.1 Oncotarget63626 www.impactjournals.com/oncotarget 7. Petersen PH, Zou K, Hwang JK, Jan YN, Zhong W. Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature. 2002; 419:929–934. 8. 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