In search of definitions: Cancer-associated fibroblasts and their markers

in International Journal of Cancer (2019)

The tumor microenvironment has been identified as one of the driving factors of tumor progression and invasion. Inside this microenvironment, cancer-associated fibroblasts (CAFs), a type of perpetually ... [more ▼]

The tumor microenvironment has been identified as one of the driving factors of tumor progression and invasion. Inside this microenvironment, cancer-associated fibroblasts (CAFs), a type of perpetually activated fibroblasts, have been implicated to have a strong tumor-modulating effect and play a key role in areas such as drug resistance. Identification of CAFs has typically been carried based on the expression of various "CAF markers", such as fibroblast activation protein alpha (FAP) and alpha smooth muscle actin (αSMA), which separates them from the larger pool of fibroblasts present in the body. However, as outlined in this Review, the expression of various commonly used fibroblast markers is extremely heterogeneous and varies strongly between different CAF subpopulations. As such, novel selection methods based on cellular function, as well as further characterizing research, are vital for the standardization of CAF identification in order to improve the cross-applicability of different research studies in the field. The aim of this review is to give a thorough overview of the commonly used fibroblast markers in the field and their various strengths and, more importantly, their weaknesses, as well as to highlight potential future avenues for CAF identification and targeting. [less ▲]

The microRNA-371~373 cluster represses colon cancer initiation and metastatic colonization by inhibiting the TGFBR2/ID1 signaling axis.

in Cancer research (2018)

The vast majority of colorectal cancer (CRC)-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key ... [more ▼]

The vast majority of colorectal cancer (CRC)-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different CRC cell lines and recently established primary cultures enriched in colon cancer stem cells (CSCs) - also known as tumor-initiating cells (TICs) - to identify genes and microRNAs (miRNAs) with regulatory functions in CRC progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, transforming growth factor beta (TGF-beta) signaling activity, and reduced expression of the miR-371~373 cluster compared to non-metastatic cultures. TGF-beta receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371~373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371~373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor-initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371~373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371~373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371~373 and ID1. Altogether, our results establish the miR-371~373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization. [less ▲]

MicroRNA regulation of hypoxia-induced tumorigenicity and metastatic potential of colon tumor-initiating cells

Doctoral thesis (2017)

The initiaton and progression of colorectal cancer (CRC), which is the second most common cause of cancer mortality in Western countries, are driven by a subpopulation of highly tumorigenic cells, known ... [more ▼]

The initiaton and progression of colorectal cancer (CRC), which is the second most common cause of cancer mortality in Western countries, are driven by a subpopulation of highly tumorigenic cells, known as cancer stem cells or tumor-initiating cells (TICs). These self-renewing TICs are, to a large extent, responsible for therapy resistance, cancer recurrence, and metastasis formation. TICs are known to extensively interact with their microenvironment and can be influenced by various extrinsic factors, such as inflammatory signaling or tumor hypoxia. Previous expression profiling studies have shown that microRNAs (miRNAs) are involved in the regulation of CRC inititation and metastatic progression. Moreover, specifc miRNAs have been identified as potential mediators of the cellular response to hypoxia. On the other hand, the molecular mechanisms that link hypoxia, miRNA expression, colon TIC regulation, and CRC progression, remain poorly understood. Thus, the main objectives of this work were to analyze the effects of hypoxia on the miRNA expression of colon TICs and to identify miRNAs that regulate metastasis initiation. In a first phase, we generated and thoroughly characterized different stable TIC-enriched spheroid cultures (SCs), both from CRC cell lines and from primary patient material. Each established SC was thereby shown to display key TIC properties, including substantial plasticity, in vitro and in vivo self-renewal capacity and, most importantly, extensive tumorigenic potential. Moreover, the individual SCs displayed increased chemoresistance capacity, compared to adherent counterpart cultures. Taken together, we could demonstrate that the spheroid system is a suitable model to study colon TICs, thereby laying the methodological foundation for the following subparts of this project. In a second step, we studied the influence of hypoxia on the miRNA expression profile of our established SCs. MiR-210-3p was thereby identified as the miRNA with the strongest response to hypoxia. Importantly, both hypoxic culture conditions and stable overexpression of miR-210 were shown to promote in vitro and in vivo self-renewal capacity of our colon TIC-enriched cultures. Moreover, by promoting lactate production and by repressing mitochondrial respiration, miR-210 was found to trigger the metabolic reprogramming of colon TICs towards a glycolytic and aggressive phenotype. Finally, we studied the role of miRNAs in the context of TIC-driven metastasis formation. By comparing primary tumor- and lymph node metastasis-derived SCs, we were able to identify the miR-371~373 cluster as an important regulator of tumorigenic and metastatic potential. Stable overexpression of the entire miR-371~373 cluster, followed by gene and protein expression analysis, enabled us to uncover the transforming growth factor beta receptor II (TGF-βRII) and the inhibitor of DNA binding 1 (Id1) as miR-371~373 cluster-responsive proteins. Most importantly, different sphere, tumor, and metastasis formation assays revealed that the miR-371~373/TGF-βRII/Id1 signaling axis regulates the self-renewal capacity and metastatic colonization potential of colon TICs. Taken together, our findings emphasize the strong plasticity of colon TICs and clearly illustrate that miRNAs can act as potent modulators of essential TIC properties. Accordingly, we could show that miR-210 and the miR-371~373 cluster are involved in metabolic reprogramming of TICs and in the regulation of metastasis formation, respectively. Altogether, our study contributes to a better understanding of the molecular mechanisms that drive TIC-induced tumor progression and may provide indications for interesting miRNA biomarker candidates and target molecules for future TIC-specific therapies. [less ▲]

Hypoxia-responsive miR-210 promotes self-renewal capacity of colon tumor-initiating cells by repressing ISCU and by inducing lactate production

in Oncotarget (2016), 7(40), 97-114

Low oxygen concentrations (hypoxia) are known to affect the cellular metabolism and have been suggested to regulate a subpopulation of cancer cells with tumorigenic properties, the so-called tumor ... [more ▼]

Low oxygen concentrations (hypoxia) are known to affect the cellular metabolism and have been suggested to regulate a subpopulation of cancer cells with tumorigenic properties, the so-called tumor-initiating cells (TICs). To better understand the mechanism of hypoxia-induced TIC activation, we set out to study the role of hypoxia-responsive miRNAs in recently established colon cancer patientderived TICs. We were able to show that low oxygen concentrations consistently lead to the upregulation of miR-210 in different primary TIC-enriched cultures. Both stable overexpression of miR-210 and knockdown of its target gene ISCU resulted in enhanced TIC self-renewal. We could validate the tumorigenic properties of miR- 210 in in vivo experiments by showing that ectopic expression of miR-210 results in increased tumor incidence. Furthermore, enhanced miR-210 expression correlated with reduced TCA cycle activity and increased lactate levels. Importantly, by blocking lactate production via inhibition of LDHA, we could reverse the promoting effect of miR-210 on self-renewal capacity, thereby emphasizing the regulatory impact of the glycolytic phenotype on colon TIC properties. Finally, by assessing expression levels in patient tissue, we could demonstrate the clinical relevance of the miR-210/ISCU signaling axis for colorectal carcinoma. Taken together, our study highlights the importance of hypoxia-induced miR-210 in the regulation of colon cancer initiation. [less ▲]