Kristel Kemper

Wnt activity defines colon cancer stem cells and is regulated by the microenvironment

Despite the presence of mutations in APC or β-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when β-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate β-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo. We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.

The AC133 Epitope, but not the CD133 Protein, Is Lost upon Cancer Stem Cell Differentiation

Colon cancer stem cells (CSC) can be identified with AC133, an antibody that detects an epitope on CD133. However, recent evidence suggests that expression of CD133 is not restricted to CSCs, but is also expressed on differentiated tumor cells. Intriguingly, we observed that detection of the AC133 epitope on the cell surface decreased upon differentiation of CSC in a manner that correlated with loss of clonogenicity. However, this event did not coincide with a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, we noted that with CSC differentiation, a change occured in CD133 glycosylation. Thus, AC133 may detect a glycosylated epitope, or differential glycosylation may cause CD133 to be retained inside the cell. We found that AC133 could effectively detect CD133 glycosylation mutants or bacterially expressed unglycosylated CD133. Moreover, cell surface biotinylation experiments revealed that differentially glycosylated CD133 could be detected on the membrane of differentiated tumor cells. Taken together, our results argue that CD133 is a cell surface molecule that is expressed on both CSC and differentiated tumor cells, but is probably differentially folded as a result of differential glycosylation to mask specific epitopes. In summary, we conclude that AC133 can be used to detect cancer stem cells, but that results from the use of this antibody should be interpreted with caution.

Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma

Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas.

Monoclonal Antibodies Against Lgr5 Identify Human Colorectal Cancer Stem Cells

In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence‐activated cell sorting‐based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+Lgr5+ cells. Similarly, primary CRC‐derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5high CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs. STEM CELLS2012;30:2378–2386

Cyclooxygenase-2 inhibition inhibits c-Met kinase activity and Wnt activity in colon cancer.

Activity of receptor tyrosine kinases (RTK) in colorectal cancer (CRC) is associated with enhanced tumor growth and a poorer prognosis. In addition, cyclooxygenase-2 (COX-2) expression contributes to tumor growth and invasion. COX-2 inhibitors exhibit important anticarcinogenic potential against CRC, but the molecular mechanism underlying this effect and the relation with RTK signaling remain the subject of intense research effort. Therefore, the rapid effects of COX-2 inhibition in CRC on the complement of all cellular kinases were investigated using a kinase substrate peptide array, Western blotting, transfection, small interfering RNA assays, and CRC cell lines. The resulting alterations in the kinome profile revealed that celecoxib, a selective COX-2 inhibitor, impairs phosphorylation of substrates for the RTKs c-Met and insulin-like growth factor receptor, resulting in decreased downstream signaling. The decrease in c-Met activation is accompanied with an increase in glycogen synthase kinase 3beta kinase activity together with a rapid increase in phosphorylation of beta-catenin. In agreement, a significant reduction of beta-catenin-T-cell factor-dependent transcription is observed both with celecoxib and selective inhibition of c-Met phosphorylation by small molecules. Hence, corepression of c-Met-related and beta-catenin-related oncogenic signal transduction seems a major effector of celecoxib in CRC, which provides a rationale to use c-Met inhibitors and celecoxib analogous to target c-Met and Wnt signaling in a therapeutic setting for patients with CRC.

Interrogating open issues in cancer precision medicine with patient-derived xenografts

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.

Intra‐ and inter‐tumor heterogeneity in a vemurafenib‐resistant melanoma patient and derived xenografts

The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAFV600E metastatic melanoma. However, resistance to such compounds represents a formidable problem. Using whole‐exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug‐resistant metastases. Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway. In addition to three independent amplifications and an aberrant form of BRAFV600E, we identified a new activating insertion in MEK1. This MEK1T55delinsRT mutation could be traced back to a fraction of the pre‐treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas. Analysis of patient‐derived xenografts (PDX) from therapy‐refractory metastases revealed that multiple resistance mechanisms were present within one metastasis. This heterogeneity, both inter‐ and intra‐tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient. In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting. Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patients melanoma.

CopywriteR: DNA copy number detection from off-target sequence data

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting ‘off-target’ sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools.

Mutations in the Ras-Raf axis underlie the prognostic value of CD133 in colorectal cancer

Purpose: High expression of cancer stem cell (CSC) marker CD133 has been used as a predictor for prognosis in colorectal cancer (CRC), suggesting that enumeration of CSCs, using CD133, is predictive for disease progression. However, we showed recently that both CD133 mRNA and protein are not downregulated during differentiation of colon CSCs, pointing to an alternative reason for the prognostic value of CD133. We therefore set out to delineate the relation between CD133 expression and prognosis. Experimental Design: A CRC patient series was studied for expression of CD133 and other CSC markers by microarray and quantitative PCR analysis. In addition, several common mutations were analyzed to determine the relation with CD133 expression. Results: CD133 mRNA expression predicted relapse-free survival in our patient series, whereas several other CSC markers could not. Moreover, no correlation was found between expression of other CSC markers and CD133. Interestingly, high CD133 expression was related to mutations in K-Ras and B-Raf, and inhibition of mutant K-Ras or downstream mitogen-activated protein kinase kinase (MEK) signaling decreases CD133 expression. In addition, an activated K-Ras gene expression signature could predict CD133 expression in our patient set as well as data sets of other tumor types. Conclusion: CD133 expression is upregulated in CRC tumors that have a hyperactivated Ras–Raf–MEK–ERK pathway and is therefore related to mutations in K-Ras or B-Raf. As mutations in either gene have been related to poor prognosis, we conclude that CD133 expression is not indicative for CSC numbers but rather related to the mutation or activity status of the Ras–Raf pathway. Clin Cancer Res; 18(11); 3132–41. ©2012 AACR.

Neoadjuvant Selective COX-2 Inhibition Down-Regulates Important Oncogenic Pathways in Patients With Esophageal Adenocarcinoma

Objectives:To evaluate the effects of neoadjuvant therapy with the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib in vitro and in patients with esophageal adenocarcinoma on COX-2 and MET expression. Summary Background Data:High COX-2 and/or MET expression levels are negative prognostic factors for adenocarcinoma of the esophagus. Nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors exert anticancer mechanisms as is evident from epidemiologic studies and from experimental models for esophageal cancer. The mechanisms and the significance of these findings in patients with adenocarcinoma of the esophagus are unknown. Methods:Esophageal adenocarcinoma cell lines were used to asses the effects in vitro. To study the clinical effects 12 patients with esophageal adenocarcinoma were included for neoadjuvant treatment (4 weeks) with celecoxib at 400 mg twice daily. Fifteen patients not receiving NSAIDs or celecoxib were included as a control. Effects were evaluated using the MTT-cell viability test, Western blot analysis, immunohistochemistry, and RT-PCR. Results:In vitro celecoxib administration resulted in decreased cell viability, increased apoptosis, and decreased COX-2 and MET expression levels. In patients, neoadjuvant treatment with celecoxib significantly down-regulated COX-2 and MET expression in the tumor when compared with the nontreated control group and when compared with pretreatment measurements. Conclusions:This is the first study to show in vitro and in patients with esophageal adenocarcinoma that selective COX-2 inhibition down-regulates COX-2 and MET expression, both important proteins involved in cancer progression and dissemination. Therefore, (neo)adjuvant therapy with celecoxib might have clinical potential for patients with esophageal adenocarcinoma.