Report18 January 2017Open Access Source DataTransparent process Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions Gabriele Picco Gabriele Picco Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Consalvo Petti Consalvo Petti Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Alessia Centonze Alessia Centonze Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Erica Torchiaro Erica Torchiaro Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Istituto Nazionale Biostrutture e Biosistemi - Consorzio Interuniversitario, Roma, Italy Search for more papers by this author Giovanni Crisafulli Giovanni Crisafulli Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Search for more papers by this author Luca Novara Luca Novara Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Search for more papers by this author Andrea Acquaviva Andrea Acquaviva Department of Computer and Control Engineering, Politecnico di Torino, Turin, Italy Search for more papers by this author Alberto Bardelli Alberto Bardelli Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Enzo Medico Corresponding Author Enzo Medico [email protected] orcid.org/0000-0002-3917-2438 Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Gabriele Picco Gabriele Picco Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Consalvo Petti Consalvo Petti Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Alessia Centonze Alessia Centonze Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Erica Torchiaro Erica Torchiaro Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Istituto Nazionale Biostrutture e Biosistemi - Consorzio Interuniversitario, Roma, Italy Search for more papers by this author Giovanni Crisafulli Giovanni Crisafulli Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Search for more papers by this author Luca Novara Luca Novara Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Search for more papers by this author Andrea Acquaviva Andrea Acquaviva Department of Computer and Control Engineering, Politecnico di Torino, Turin, Italy Search for more papers by this author Alberto Bardelli Alberto Bardelli Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Enzo Medico Corresponding Author Enzo Medico [email protected] orcid.org/0000-0002-3917-2438 Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy Department of Oncology, University of Torino, Candiolo, Torino, Italy Search for more papers by this author Author Information Gabriele Picco1,2, Consalvo Petti1,2, Alessia Centonze2, Erica Torchiaro1,3, Giovanni Crisafulli1, Luca Novara1, Andrea Acquaviva4, Alberto Bardelli1,2 and Enzo Medico *,1,2 1Candiolo Cancer Institute – FPO IRCCS, Candiolo, Torino, Italy 2Department of Oncology, University of Torino, Candiolo, Torino, Italy 3Istituto Nazionale Biostrutture e Biosistemi - Consorzio Interuniversitario, Roma, Italy 4Department of Computer and Control Engineering, Politecnico di Torino, Turin, Italy *Corresponding author. Tel: +39 011 9933 234; Fax: +39 011 9933225; E-mail: [email protected] EMBO Mol Med (2017)9:293-303https://doi.org/10.15252/emmm.201606773 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In colorectal cancer (CRC), WNT pathway activation by genetic rearrangements of RSPO3 is emerging as a promising target. However, its low prevalence severely limits availability of preclinical models for in-depth characterization. Using a pipeline designed to suppress stroma-derived signal, we find that RSPO3 "outlier" expression in CRC samples highlights translocation and fusion transcript expression. Outlier search in 151 CRC cell lines identified VACO6 and SNU1411 cells as carriers of, respectively, a canonical PTPRK(e1)-RSPO3(e2) fusion and a novel PTPRK(e13)-RSPO3(e2) fusion. Both lines displayed marked in vitro and in vivo sensitivity to WNT blockade by the porcupine inhibitor LGK974, associated with transcriptional and morphological evidence of WNT pathway suppression. Long-term treatment of VACO6 cells with LGK974 led to the emergence of a resistant population carrying two frameshift deletions of the WNT pathway inhibitor AXIN1, with consequent protein loss. Suppression of AXIN1 in parental VACO6 cells by RNA interference conferred marked resistance to LGK974. These results provide the first mechanism of secondary resistance to WNT pathway inhibition. Synopsis In colorectal cancer (CRC), translocations of the RSPO3 gene, leading to its expression as a fusion transcript, pinpoint a therapeutically actionable mechanism of Wnt pathway activation. RSPO3 "outlier" expression analysis allowed identification of CRC cases featuring RSPO3 rearrangements. CRC cell lines with RSPO3 rearrangements were identified and found to be sensitive to Wnt ligand deprivation induced by PORCN inhibition. Secondary resistance to WNT pathway blockade was associated with loss-of-function mutations in the AXIN1 gene. Introduction Most colorectal cancers display aberrant activation of the WNT pathway, leading to stabilization and nuclear translocation of β-catenin, a key transcriptional regulator controlling stem cell maintenance, proliferation, and differentiation (Krausova & Korinek, 2014). In a molecular survey carried out by The Cancer Genome Atlas, 93% of CRCs have been found to carry a genetic alteration in at least one of 16 WNT pathway genes, defining this pathway as a major driver of CRC (Cancer Genome Atlas, 2012). In particular, loss of function of the WNT pathway suppressor APC accounts for 70% of all CRCs. APC alterations typically occur at early steps of the colorectal adenoma–carcinoma sequence (Powell et al, 1992; Morin et al, 1997), a typical feature of "trunk" genetic events, present in all cancer cells and therefore therapeutically attractive (Swanton, 2012). Accordingly, established CRCs were found to critically depend on APC mutation-driven enhanced WNT signaling, even in the presence of additional cancer-driving mutations (Dow et al, 2015). Genomic rearrangements involving the RSPO2 and RSPO3 genes have been found to provide an alternative mechanism of aberrant WNT pathway activation in CRC (Seshagiri et al, 2012). These genes encode secreted proteins, R-spondins, that synergize with WNT ligands to promote β-catenin signaling (de Lau et al, 2011). RSPO2 and RSPO3 translocations are mutually exclusive with APC mutations and lead to aberrant expression of fusion transcripts in which the 5′ portion, upstream from the RSPO coding sequence, is typically contributed by the highly expressed EIF3E and PTPRK genes, respectively (Seshagiri et al, 2012). In the case of the PTPRK-RSPO3 translocation, recent studies in patient-derived xenografts (PDXs) carrying the fusion transcript demonstrated that inhibition of WNT ligand secretion by porcupine blockade, or direct targeting of RSPO3 by antibodies, markedly inhibits tumor growth, promoting loss of cancer stem cell functions and differentiation (Chartier et al, 2016; Madan et al, 2016; Storm et al, 2016). These results highlight the clinical relevance of targeting RSPO3 rearrangements in CRC patients. As for all pathway-targeted therapies, availability of experimental models is crucial to characterize the dependency on pathway activators and to explore possible mechanisms of release from such dependency (Misale et al, 2014; Rosa et al, 2014). Indeed, mechanisms of acquired resistance to WNT pathway inhibition are currently unexplored. In this view, availability of established CRC cell lines spontaneously carrying RSPO3 fusions would provide a valuable resource. We recently reported a large collection of 151 CRC cell lines and associated global gene expression profiles, reliably representing the molecular heterogeneity of CRC (Medico et al, 2015). We have also shown that expression-based outlier analysis within this compendium leads to identification of therapeutically actionable oncogenic drivers (Medico et al, 2015). Considering that, in RSPO3 rearrangements, a strong promoter is placed upstream of the RSPO3 coding sequence, we hypothesized that searching for outlier RSPO3 expression might pinpoint cases carrying the respective translocation. This hypothesis was tested in RNA-seq-based expression data generated by the TCGA, checking the presence of the RSPO3 fusion in outlier cases. Outlier expression analysis was then extended to the 151 CRC cell lines, leading to the identification of two unique lines carrying a RSPO3 fusion. We employed these cell lines to investigate addiction to WNT pathway activation mediated by PTPRK-RSPO3 translocations, modeling primary sensitivity and acquired secondary resistance. Results and Discussion Identification of RSPO3 rearrangements in CRC samples by combined stroma/transcript expression analysis PTPRK-RSPO3 rearrangements typically lead to aberrant expression of fusion transcripts by colorectal cancer cells that normally do not express RSPO3 (Sato et al, 2009; Seshagiri et al, 2012). Therefore, in principle, searching for CRC samples displaying high RSPO3 mRNA levels should pinpoint tumors bearing RSPO3 fusions. However, stromal cells have been identified as a source for RSPO3 expression in the intestine (Kabiri et al, 2014). To verify the stromal origin of RSPO3 transcripts in CRC, we exploited the fact that in CRC patient-derived xenografts (PDXs), human stroma is substituted by mouse stroma (Isella et al, 2015). Analysis of our previous species-specific analysis of RNA-seq data from CRC PDXs (Isella et al, 2015) revealed that the fraction of RSPO3 transcripts of mouse origin is 99.9%. As a consequence, it is expected that in a human CRC sample, RSPO3 mRNA levels are correlated with the richness of stroma that we found to be reliably approximated by a transcriptional cancer-associated fibroblast (CAF) score (Isella et al, 2015). We therefore analyzed the correlation between the CAF score and RSPO3 mRNA levels in a 450-sample human CRC RNA-seq expression dataset that we previously assembled from TCGA (Isella et al, 2015). As shown in Fig 1A, RSPO3 levels were highly correlated with the CAF score (Pearson correlation = 0.76), confirming that the expression of this gene in tumor samples is mostly due to stromal cells. However, among the samples expressing high RSPO3 levels (red rectangle in Fig 1A), some had low CAF score, suggesting a possible overexpression by epithelial cancer cells. Similar results were also obtained using additional stromal scores, namely the endothelial score and the leukocyte score (Appendix Fig S1). We therefore selected all cases with RSPO3 expression Z-score > 2 and searched for RSPO3 fusions in the corresponding TCGA RNA-seq data. A canonical PRPTK(ex1)-RSPO3(ex2) fusion transcript was detected in 6 out of the 14 selected samples (43%). To test whether RSPO3 fusions are enriched in samples with low stroma, for each of the 450 TCGA samples, we subtracted the CAF score from the RSPO3 expression value and found that in a subset of samples, presenting low CAF score values and high RSPO3 expression, this delta value raises to Z-score levels usually observed for outlier genes (Fig 1B). The relationship between RSPO3 levels and CAF score was further validated in an independent 2140-sample CRC microarray dataset, highlighting the presence of samples with high RSPO3 expression not justified by abundant stroma (Appendix Fig S2). In principle, RSPO3 fusions are expressed only by cancer cells; therefore, outlier samples characterized by low CAF score should be enriched in fusion transcripts. To test this hypothesis, the 14 RSPO3 overexpressing samples described above were subdivided in two groups, respectively, having a positive and a negative delta (RSPO3-CAF score). Notably, the six RSPO3 fusions were exclusively present in the positive delta group, also corresponding to delta Z-score values higher than 2.5 (Fig 1B, Appendix Table S1). To verify whether additional RSPO3 fusions were present in samples not identified by the above approach, we extended the search for RSPO3 fusions to 12 additional TCGA RNA-seq samples expressing lower levels of RSPO3. In particular, for any expression level, we selected samples with the lowest CAF score, so that at least a fraction of the RSPO3 reads could theoretically derive from epithelial cells (Appendix Fig S3). None of these samples was found to carry a RSPO3 fusion, suggesting that our approach saturated the dataset and captured all the samples carrying RSPO3 rearrangements. Samples with lower levels were not explored because the limited number of expected RNA-seq reads covering RSPO3 would anyway not allow detection of reads covering a fusion transcript. Cross-check with an available pan-cancer database of fusion transcripts in TCGA samples (http://www.tumorfusions.org; Yoshihara et al, 2015) revealed that no additional RSPO3 fusions were detected in the 450-sample TCGA dataset used for our analysis. Figure 1. Identification of RSPO3 rearrangements in CRC samples and cell lines by outlier expression analysis Scatter plot displaying the correlation between RSPO3 expression (x-axis) and CAF score (y-axis). The red box highlights samples with high RSPO3 expression and variable CAF score. Empty dots indicate samples selected for fusion analysis. Empty dots with red border indicate samples with high RSPO3 levels and medium-low CAF score. Dot plots displaying the distribution of Z-score values for RSPO3 alone (left panel) and RSPO3 minus CAF score (right panel) in the 450-sample TCGA dataset. Red dots indicate RSPO3 fused samples. Empty dots indicate analyzed samples that do not carry fusions in the RSPO3 gene. Scatter plot displaying RSPO3 expression levels (Log2 signal, y-axis) in 151 CRC cell lines (left). Right images represents SNU1411 and VACO6 cell lines phase-contrast micrographs. Canonical in-frame gene fusion between exon 1 of PTPRK and exon 2 of the RSPO3 gene revealed by RNA-seq analysis of VACO6 cells. Novel in-frame gene fusion between exon 13 of PTPRK and exon 2 of the RSPO3 gene revealed by RNA-seq analysis of SNU1411 cells. Download figure Download PowerPoint These results show that the presence of RSPO3 rearrangements in CRC can be anticipated by high RSPO3 mRNA levels not justified by abundant stroma. Transcriptional outlier analysis identifies CRC cell lines carrying canonical and novel PTPRK-RSPO3 rearrangements To search for established cell lines carrying RSPO3 rearrangements, we analyzed RSPO3 expression levels in our previously described compendium of 151 CRC cell lines (Medico et al, 2015). While, as expected, RSPO3 expression was low or absent in most cells, two lines, VACO6 and SNU1411, were found to markedly overexpress RSPO3 (Fig 1C). Notably, both cell lines are wild type for APC, but have a different molecular makeup: VACO6 are microsatellite instable and BRAF mutated (V600E); SNU1411 are microsatellite stable and KRAS mutated (G12C). These mutations render both cells insensitive to EGFR-blockade by cetuximab (Medico et al, 2015) and therefore attractive models for alternative targeted therapy approaches. The two lines have also different in vitro growth properties (Fig 1C, photo inserts): while SNU1411 form adherent colonies (Ku et al, 2010), VACO6 cells, established from a poorly differentiated cecal cancer, grow as aggregates in suspension (McBain et al, 1984). RNA-seq analysis of the VACO6 and SNU1411 transcriptomes revealed a canonical PRPTK(ex1)-RSPO3(ex2) fusion transcript in VACO6 (Fig 1D) and a novel PRPTK(ex13)-RSPO3(ex2) fusion transcript in SNU1411 cells (Fig 1E). RNA-seq results were further confirmed by PCR followed by Sanger sequencing (Appendix Fig S4). The absence of reads covering exon 1 of RSPO3 confirmed that all RSPO3 transcripts detected in these cell lines originate from the fused transcript. These results highlight VACO6 and SNU1411 cell lines as unique cell line models to characterize addiction to WNT pathway activation by rearranged RSPO3 not only in vivo, but also in vitro, in the absence of supporting stroma. Cell lines carrying RSPO3 fusion transcripts are sensitive to porcupine inhibition in vitro and in vivo The vast majority of CRC are affected by loss-of-function mutations in components of the destruction complex (e.g., APC) leading to accumulation of β-catenin and constitutive activation of Wnt target genes. RSPO3 instead promotes WNT pathway activation by binding the LGR4/5 protein and neutralizing RNF43-mediated degradation of LRP5/6 receptor, enhancing therefore the activity of WNT ligands (de Lau et al, 2014). Therefore, pharmacological blockade of WNT ligand secretion could be an effective strategy to target RSPO3-overexpressing cancer cells (Madan & Virshup, 2015). To this aim, we considered the small molecule LGK974 that specifically inhibits the porcupine (PORCN) acyltransferase, thus abrogating posttranslational processing and secretion of WNT ligands (Krausova & Korinek, 2014). LGK974 is currently being tested in humans, in phase 1 trials (Liu et al, 2013). VACO6 and SNU1411 cells were tested for in vitro dose–response to LGK974 and found to be both exquisitely sensitive, with IC50 values below 50 nM (Fig 2A). As a control, HCT116 cells, that do not carry RSPO3 rearrangements, were insensitive to PORCN inhibition (IC50 > 5 μM). As shown in Fig 2B, both cell lines responded to LGK974 with marked apoptotic cell death and downregulation of the WNT pathway, evaluated by quantitative reverse transcription PCR (qRT–PCR) analysis of the WNT target gene AXIN2 (Drost et al, 2015; Jho et al, 2002; see Materials and Methods). To evaluate in vivo sensitivity of VACO6 and SNU1411 cells to WNT pathway inhibition, immunocompromised mice were xenotransplanted and treated with LGK974 or vehicle for 4 weeks. Xenotransplants of both cell lines responded to LGK974 with sustained growth inhibition (> 90%) and tumor stabilization (Fig 2C and D). Accordingly, tumors explanted at the end of the treatment displayed dramatic reduction in proliferating cells, and mucinous differentiation (Fig 2E and F), confirming that the in vivo response of both cell lines to WNT blockade phenocopies the described differentiation and growth arrest observed in CRC patient-derived xenografts (Storm et al, 2016). Altogether, these results show that both CRC cell lines carrying RSPO3 fusions are addicted to WNT signaling and sensitive to the porcupine inhibitor LGK974 in vitro and in vivo. Moreover, both models completely recapitulate morphofunctional traits of response previously described for RSPO3 blockade (Madan & Virshup, 2015; Storm et al, 2016). This is particularly interesting for SNU1411 cells with the noncanonical RSPO3 fusion, in which the long upstream coding sequence from PTPRK does not seem to lessen the pathological activation of WNT pathway driven by aberrant RSPO3 expression. Figure 2. VACO6 and SNU1411 cells are sensitive to the porcupine inhibitor LGK974 in vitro and in vivo A. Dose–response cell viability assay on VACO6, SNU1411, and HCT116 cells treated with LGK974. Data are expressed as average ± SD of six technical replicates from one representative experiment. B. Effect of 1 μM LGK974 on apoptosis and WNT pathway activity in VACO6 and SNU1411 cells. Upper panel: percentage of viable cells (DAPI-negative & Annexin-negative; y-axis) after 96 h in control medium or 1 μM LGK974; means ± SDs from triplicate experiments. Asterisks represent significant differences measured by Student's t-test (two-sided), **P < 0.05; ***P < 0.001. VACO6 (LGK974 versus CTRL), P = 3.88E-05; SNU1411 (LGK974 versus CTRL), P = 5.54E-03. Lower panel: qRT–PCR evaluation of AXIN2 mRNA after treatment with vehicle or 1 μM LGK974 for 24 h, as indicated. Data are expressed as means ± RMSE of three technical replicates from one representative experiment measured by Student's t-test (two-sided), **P < 0.05 (0.006); ***P < 0.001). VACO6 (LGK974 versus CTRL), P = 1.72E-03; SNU1411 (LGK974 versus CTRL), P = 6.12E-05. C, D. Tumor growth curves of PDXs from VACO6 and SNU1411 xenografts treated for 28 days with LGK974 (5 mg/kg; red line) or with vehicle (black line). Number of replicates: 6 or 7, as indicated in the panels. Error bars indicate standard error of the mean (SEM) values. E, F. Micrographs of representative xenograft tumors derived from VACO6 (E) and SNU1411 (F) explanted at the end of treatment with vehicle or LGK974, stained with Ki67 or PAS, as indicated. Scale bar, 50 μm. Download figure Download PowerPoint AXIN1 frameshift deletions confer acquired resistance to WNT pathway inhibition in RSPO3-addicted cells VACO6 cells, carrying the most common RSPO3 fusion, were subjected to long-term treatment with incremental doses of LGK974 (see Materials and Methods), which was found to generate secondary resistance within 3 months. In the meantime, parental cells were maintained in culture without drug as a control. A viability assay demonstrated that selected cells were completely resistant to LGK974 (IC50 > 10 μM), while control parental cells maintained their sensitivity (Fig 3A). Copy number analysis based on exome sequencing of LGK974-resistant and parental VACO6 cells only highlighted minor changes of no clear functional meaning: trisomy of chromosome 8 and heterozygous deletion from 13q21.39 to 13q31.1. Moreover, a series of additional indels/mutations with high allelic frequency were detected (Appendix Table S2). In the context of a MSI cell line, a large set of mutations at low allelic frequency is expected as a consequence of genetic drift during expansion. However, in this case, the number of concurrent mutations at high allelic frequency points to the occurrence of a separated clone, pre-existing to the selection process. Interestingly, exome sequencing revealed that LGK974-resistant VACO6 (VACO6R) cells diverged from the parental population by the presence of two single-base deletions in the AXIN1 coding sequence (Fig 3B, Appendix Fig S5), respectively, at position p.G265fs* (33 supporting reads) and p.V835fs* (90 supporting reads). Both deletions are reported in the COSMIC database (Forbes et al, 2015) as cancer-related somatic variants (COSM1609260 and COSM2920077) and the first one is predicted to be a truncating alteration (Cerami et al, 2012). Indeed, both mutations, detected as heterozygous in VACO6R cells, induce a frameshift in the coding sequence, which is compatible with a functionally homozygous AXIN1 loss of function. Accordingly, Western blot analysis showed a dramatic reduction in AXIN1 protein expression in VACO6R cells (Fig 3C). Figure 3. AXIN1 loss confers acquired resistance to PORCN inhibition in VACO6 cell lines Cell viability assay of VACO6 and VACO6R cells after 6 days of treatment with LGK974. Data are expressed as average ± SD of six technical replicates from one representative experiment. Representative chromatograms of Sanger-sequenced PCR products confirming the two concurrent AXIN1 frameshift deletions (FS) in VACO6R cells. Western blot showing loss of the AXIN1 protein in VACO6R cells. Bar graph displaying relative mRNA levels of the WNT pathway target gene AXIN2, measured by qRT–PCR, in VACO6 and VACO6R cells. Two-sided Student's t-test, *P < 0.05, P = 1.42E-02. Data are expressed as means ± SD of three technical replicates from one representative experiment. Western blot showing downregulation of the AXIN1 protein in VACO6 cells by RNAi. ATP-based assay measuring viability, after 6 days of LGK974 treatment, of VACO6 cells transiently transfected with control siRNA or with AXIN1 siRNA, as indicated. Data are expressed as average ± SD of six technical replicates from one representative experiment. Schematic representation of (i) how RSPO3 overexpression supports WNT-β-catenin signaling, (ii) how porcupine inhibition by LGK974 blocks β-catenin, and (iii) how AXIN1 loss restores β-catenin signaling in the presence of LGK974. Source data are available online for this figure. Source Data for Figure 3 [emmm201606773-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint To verify whether the two AXIN1 mutations co-existed in the same resistant clone, we isolated 14 independent clones from VACO6R cells by limiting dilution. All clones displayed complete loss of AXIN1 protein, as previously observed for VACO6R (Appendix Fig S6). Accordingly, we found that both frameshift mutations were present in all clones, each of them being heterozygous (Appendix Fig S6), which suggests that the vast majority of VACO6R cells emerged from one resistant subclone in which the two AXIN1 alleles were independently inactivated. The AXIN1 protein acts as a scaffold for the destruction complex, in which GSK-3β phosphorylates β-catenin, leading to its ubiquitination and degradation. Therefore, loss of AXIN1 leads to stabilization of β-catenin and aberrant activation of the WNT pathway (MacDonald et al, 2009). Accordingly, VACO6R cells displayed enhanced mRNA expression of the WNT target AXIN2 (Fig 3D). AXIN1 and AXIN2 are considered to be functionally redundant (Chia & Costantini, 2005); however, AXIN2 is not always able to compensate for AXIN1 knockdown (Figeac & Zammit, 2015). Indeed, in the case of VACO6R cells, the observed AXIN2 upregulation does not effectively counteract the complete loss of AXIN1, which results in overall enhancement of WNT signaling. To validate AXIN1 loss as a mechanism of secondary resistance to WNT pathway inhibition, we downregulated AXIN1 by RNA interference in parental VACO6 cells. Transient transfection of siRNAs against AXIN1 severely reduced AXIN1 protein expression in VACO6 parental cells (Fig 3E) and rendered them markedly resistant to LGK974 (Fig 3F). We verified by qRT–PCR that the RSPO3 fusion transcript was still expressed in VACO6R cells (Fig EV1A). Accordingly, in the mirror experiment, VACO6R cells were transduced with wild-type AXIN1 to levels comparable to parental cells (VACO6RAXIN1FL; Fig EV1B), which led to reduction in WNT signaling and reversion of resistance to LGK974 (Fig EV1C and D). These results further confirmed the causative role of AXIN1 loss in resistance to LGK974. Click here to expand this figure. Figure EV1. Re-expression of the AXIN1 coding sequence re-sensitizes VACO6R cells to PORCN inhibition qRT–PCR evaluation of PTPRK-RSPO3 fusion transcript expression in VACO6, VACO6R, and HCT116 cells, as indicated. Data are expressed as means ± SD of three technical replicates from one representative experiment. Western blot showing AXIN1 protein expression in VACO6R, VACO6, and VACO6RAXIN1FL cells, as indicated. Flow cytometry analysis of the GFP signal in VACO6, VACO6R, and VACO6RAXIN1FL cells, all transduced with the 7xTcf-eGFP WNT reporter. ATP-based assay measuring viability, after 6 days of LGK974 treatment, of parental VACO6, VACO6R, and VACO6RAXIN1FL cells, as indicated. Data are expressed as average ± SD of six technical replicates. Source data are available online for this figure. Download figure Download PowerPoint Genetic alterations leading to AXIN1 loss of function have been described in colon and other cancer types, albeit at low frequency (Satoh et al, 2000; Dahmen et al, 2001; Laurent-Puig et al, 2001; Wu et al, 2001; Cancer Genome Atlas, 2012; Forbes et al, 2015). In CRC, AXIN1 alterations typically occur between exon 1 and 5, where the binding domains for APC, GSK3, and β-catenin are located (Salahshor & Woodgett, 2005). In cell lines and transgenic mice, overexpression of AXIN1 leads to increased β-catenin degradation and attenuation of WNT signaling, supporting its tumor suppressor activity (Kishida et al, 1998; Hsu et al, 2001). To verify whether AXIN1 loss confers resistance to additional WNT pathway inhibitors, we tested in vitro on VACO6 and VACO6R cells the alternative porcupine inhibitor WNT-C59 and the tankyrase inhibitor XAV939. While both WNT-C59 and XAV939 were effective on VACO6 parental cells, they had no effect on VACO6R cells (Appendix Fig S7A and B). To evaluate the pathway specificity of resistance in VACO6R, we assessed their sensitivity to two chemotherapeutic agents commonly used to treat CRC patients, the antimetabolite 5-FU and the topoisomerase-I inhibitor SN38, and to Pevonedistat, a NEDD-8 inhibitor preclinically validated in CRC, to which parental VACO6 cells are markedly sensitive (Picco et al, 2017). We found that parental and VACO6R cells display the same pattern of response to all three compounds (Appendix Fig S7C–E), indicating that AXIN1 loss confers
The copper(II) complex A0 induces non-apoptotic programmed cell death in human HT1080 fibrosarcoma cells but not in normal fibroblasts (J Med Chem, 50(8):1916–1924, 2007). While typical apoptotic features, such as caspase-3 activation or nuclear fragmentation, are evident in cisplatin-treated cells, they are absent in A0-dependent cell death. In contrast, the latter process is hallmarked by the development of huge vacuoles originating from endoplasmic reticulum (Histochem Cell Biol 126(4):473–482, 2006), a feature consistent with the newly described type of cell death named paraptosis (PNAS 97(26):14376–14381, 2000). Consistently, in a panel of human cancer cells there is no correlation between the sensitivities to A0 and cis-platin. In the same panel, paraptosis-like cell death is observed in all the A0-sensitive cell lines. Moreover, the copper complex kills cisplatin-sensitive cells (HT1080 and ovarian carcinoma 2008) as well as their cisplatin-resistant counterparts (C13* cells and the newly established HT1080PTR line) with comparable potencies. The different activity spectrum between A0 and cisplatin suggests distinct mechanisms of action for the two drugs. In agreement with this hypothesis, a whole-genome expression analysis, performed in HT1080 cells, showed that the transcriptional response evoked by the two drugs is poorly overlapping. A0 induces genes involved in oxidative- and endoplasmic reticulum-stress (ER stress), while cisplatin increases the expression of typical p53 targets. Moreover, A0 strongly induces metal responsive genes, as well as HSPs, chaperones and other genes involved in the Unfolded Protein Response (UPR). The validation of the microarray results by qRT PCR and Western Blot confirms that A0, but not cisplatin, activates two pathways of the UPR. In particular, IRE1 mRNA is up-regulated, resulting in the increased abundance of the spliced form of XBP1 mRNA that encodes for the active transcription factor. Moreover, the translation initiator complex subunit eIF2alpha is rapidly phosphorylated, with the consequent attenuation of protein synthesis and the concomitant preferential translation of the pro-death ER stress responsive proteins ATF4, CHOP and GADD34. In conclusion, A0 kills sensitive cancer cells through the triggering of ER stress, inducing a paraptotic process. Therefore, the copper complex may constitute a novel device to overcome apoptosis resistance.
