IL1R1+ cancer-associated fibroblasts drive tumor development and immunosuppression in colorectal cancer.

Fibroblasts have a considerable functional and molecular heterogeneity and can play various roles in the tumor microenvironment. Here we identify a pro-tumorigenic IL1R1+, IL-1-high-signaling subtype of fibroblasts, using multiple colorectal cancer (CRC) patient single cell sequencing datasets. This subtype of fibroblasts is linked to T cell and macrophage suppression and leads to increased cancer cell growth in 3D co-culture assays. Furthermore, both a fibroblast-specific IL1R1 knockout and IL-1 receptor antagonist Anakinra administration reduce tumor growth in vivo. This is accompanied by reduced intratumoral Th17 cell infiltration. Accordingly, CRC patients who present with IL1R1-expressing cancer-associated-fibroblasts (CAFs), also display elevated levels of immune exhaustion markers, as well as an increased Th17 score and an overall worse survival. Altogether, this study underlines the therapeutic value of targeting IL1R1-expressing CAFs in the context of CRC.

Stromal-derived IGF2 promotes colon cancer progression via paracrine and autocrine mechanisms.

The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-ß (TGFß) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFß/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFß to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFß was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.

Autocrine WNT2 signaling in fibroblasts promotes colorectal cancer progression.

The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/ß-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/ß-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/ß-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/ß-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.

IGFBP7, a novel tumor stroma marker, with growth-promoting effects in colon cancer through a paracrine tumor-stroma interaction.

The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-ß (TGFß) pathway activation were induced in CAFs. The putative TGFß-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.

Neurogenic differentiation of amniotic fluid stem cells.

In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.

Proliferation potential of human amniotic fluid stem cells differently responds to mercury and lead exposure.

There are considerable gaps in our knowledge on cell biological effects induced by the heavy metals mercury (Hg) and lead (Pb). In the present study we aimed to explore the effects of these toxicants on proliferation and cell size of primary human amniotic fluid stem (AFS) cells. Monoclonal human AFS cells were incubated with three dosages of Hg and Pb (single and combined treatment; ranging from physiological to cytotoxic concentrations) and the intracellular Hg and Pb concentrations were analyzed, respectively. At different days of incubation the effects of Hg and Pb on proliferation, cell size, apoptosis, and expression of cyclins and the cyclin-dependent kinase inhibitor p27 were investigated. Whereas we found Hg to trigger pronounced effects on proliferation of human AFS cells already at low concentrations, anti-proliferative effects of Pb could only be detected at high concentrations. Exposure to high dose of Hg induced pronounced downregulation of cyclin A confirming the anti-proliferative effects observed for Hg. Co-exposure to Hg and Pb did not cause additive effects on proliferation and size of AFS cells, and on cyclin A expression. Our here presented data provide evidence that the different toxicological effects of Pb and Hg on primary human stem cells are due to different intracellular accumulation levels of these two toxicants. These findings allow new insights into the functional consequences of Pb and Hg for mammalian stem cells and into the cell biological behavior of AFS cells in response to toxicants.

Nucleocytoplasmic localization of p70 S6K1, but not of its isoforms p85 and p31, is regulated by TSC2/mTOR.

The tuberous sclerosis complex gene 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway controls many cellular functions via phosphorylation of ribosomal protein S6 kinases (S6Ks). Alternative splicing and translation generate three S6K1 proteins. Although nuclear and cytoplasmic S6K targets are known, the nucleocytoplasmic localization of the S6K1 proteins has not been comparatively elucidated so far. We show that in primary fibroblasts p85 S6K1 is cytoplasmic, p70 can be found in both compartments and p31 is exclusively nuclear. As already known for p70 and p85, our data suggest that p31 is also a target of mTOR-mediated phosphorylation. Blocking mTOR kinase activity via rapamycin and its activation in TSC2(-/-) cells and via TSC2 small interfering RNAs revealed that it regulates the localization of p70, but not of p85 and p31. The mTOR-dependent phosphorylation of p70 S6K1 at T389 is essential for its nuclear localization and exclusively hyperphosphorylated p70 S6K1 can be found in the nucleus. We further demonstrate this mTOR-controlled p70 S6K1 localization to be growth factor dependent. During the cell-cycle phosphorylation and nuclear localization of p70 S6K1 occur in mid G1 phase. We report that the different S6K1 proteins exhibit different nucleocytoplasmic localizations and that the TSC2/mTOR cascade not only regulates p70 S6K1 activity, but also its localization. These findings provide new important insights into the temporal and spatial dynamics of TSC2/mTOR/S6K regulation.

Embryoid body formation of human amniotic fluid stem cells depends on mTOR.

Human amniotic fluid stem cells (hAFSCs) harbor high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells. The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the principal step in the differentiation of pluripotent embryonic stem cells. Using c-Kit-positive hAFSC lines, we show here that these stem cells harbor the potential to form EBs. As part of the two kinase complexes, mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signaling pathway, which is involved in the regulation of cell proliferation, growth, tumor development and differentiation. Blocking intracellular mTOR activity through the inhibitor rapamycin or through specific small interfering RNA approaches revealed hAFSC EB formation to depend on mTORC1 and mTORC2. These findings demonstrate hAFSCs to be a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development and identify the mTOR pathway to be essential for this process.

CDKs as therapeutic targets for the human genetic disease tuberous sclerosis?

The tuberous sclerosis gene 2 product tuberin is an important regulator of the mammalian target of rapamycin (mTOR). In addition, tuberin is known to bind to the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) and to regulate its stability and localization via mTOR-independent mechanisms. Recently, evidence has been provided that tuberin also affects p27 localization via regulating mTOR's potential to activate the serum- and glucocorticoid-inducible kinase (SGK1) to phosphorylate p27. Taken together, these findings strengthen the argument that besides mTOR-inhibitors, such as rapamycin analogues, p27 and CDKs could also be considered targets for hamartoma therapeutics in tuberous sclerosis.

Skp2 inversely correlates with p27 and tuberin in transformed cells.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) is a major gatekeeper of the mammalian cell cycle progression known to be regulated by both, its subcellular localization and its degradation. To allow entrance into S phase and thereby mammalian cell cycle progression p27 must be degraded by a skp2-containing E3 ubiquitin ligase whose task is to target p27 for degradation by the proteasome. The tumor suppressor gene product tuberin directly binds to p27 and protects it from degradation via skp2. Whereas, p27 and tuberin are known to be localized to both, the cytoplasm and the nucleus, the localization of skp2 remained elusive. Here we demonstrate that skp2 is a cytoplasmic and nuclear protein. In addition we found an inverse correlation of the endogenous protein levels of skp2 with p27 and tuberin in different transformed cells and under different growth conditions. These data allow new important insights into this molecular network of cell cycle control.

Tuberin, p27 and mTOR in different cells.

Mutations in the genes TSC1 or TSC2 cause the autosomal dominantly inherited tumor suppressor syndrome tuberous sclerosis, which is characterized by the development of tumors, named hamartomas, in different organs. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component. Both, hamartin and tuberin have been implicated in the control of the cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR) a regulator of the p70 ribosomal protein S6 kinase (p70S6K) and its target the ribosomal protein S6. The tuberin/hamartin complex was shown to protect p27 from protein degradation. Within the mTOR signaling pathway tuberin harbors GTPase activating (GAP) potential toward Rheb, which is a potent regulator of mTOR. In this study, we have analyzed the protein levels of tuberin, p27, cyclin D1, mTOR and phospho mTOR Ser2448 (activated mTOR), S6 and phospho S6 Ser240/244 (activated S6) and as controls alpha-tubulin and topoisomerase IIbeta, in ten different cells, including primary normal cells, immortalized and transformed cell lines.

Human amniotic fluid stem cells: a new perspective.

The discovery of amniotic fluid stem cells initiated a new and very promising field in stem cell research. In the last four years amniotic fluid stem cells have been shown to express markers specific to pluripotent stem cells, such as Oct-4. Due to their high proliferation potential, amniotic fluid stem cell lineages can be established. Meanwhile, they have been shown to harbor the potential to differentiate into cells of all three embryonic germ layers. It will be a major aim for the future to define the potential of this new source of stem cells for therapies related to specific diseases.

Ras mediates cell survival by regulating tuberin.

Mutational activation of Ras promotes oncogenesis by controlling cell cycle regulation and cell survival. Ras-mediated activation of both, the PI3K/AKT pathway and the MEK/ERK pathway, can trigger downregulation of the function of tuberin to block the activities of mTOR and p70S6K. Here we demonstrate that Ras-induced cell survival is accompanied by upregulation of p70S6K activity. Ras harbors the potential to negatively affect tuberin-induced apoptosis and p70S6K inactivation. These effects of Ras were found to depend on its potential to regulate the MEK/ERK pathway. Experiments using tuberin-negative fibroblasts revealed that the potential of Ras to counteract apoptosis depends on functional tuberin. Taken together, we provide evidence that the function of Ras to trigger inactivation of tuberin plays a major role in the regulation of cell survival upon mutational activation of the oncogene Ras. This is the first description of a functional interaction between the tumor suppressor tuberin and the oncogene Ras in regulating apoptosis.

Intratumoral IGF-I protein expression is selectively upregulated in breast cancer patients with BRCA1/2 mutations.

BRCA1/2 mutations predispose to early onset breast and ovarian cancers. The phenotypic expression of mutant alleles, however, is thought to be modified by factors that are also involved in the pathogenesis of sporadic breast cancer. One such protein is IGF-I, one of the strongest mitogens to breast cancer cells in vitro. We have utilized immunohistochemistry to compare the intratumoral IGF-I and IGF-I receptor (IGF-IR) protein expression in 57 BRCA1/2 mutation carriers and 102 matched breast cancer patients without a family history in a nested case-control study. BRCA1 silencing by siRNA was used to investigate the effect of BRCA mutations on IGF-I protein expression. IGF-I protein expression was detected in tumoral epithelium and surrounding stroma, and was significantly upregulated in tumors of BRCA mutation carriers when compared with matched sporadic tumors (epithelial: 87.7% vs 61.8%, P=0.001; stromal: 73.7% vs 34.3%, P<0.001). By contrast, IGF-IR protein expression was confined to malignant epithelium and was unchanged in mutation carriers (52.6% vs 39.2%, P=0.310). While in mutation carriers IGF-IR protein expression was significantly correlated with both epithelial (P=0.003) and stromal IGF-I (P=0.02), this association was less pronounced in sporadic breast cancer (P=0.02 respectively). siRNA-mediated downregulation of BRCA1 in primary human mammary gland cells triggered upregulation of endogenous intracellular IGF-I in vitro. The increased intratumoral IGF-I protein expression in BRCA mutation carriers suggests an involvement of the IGF-I/IGF-IR axis in the biological behavior of breast cancers in this population and could define a potential therapeutic target.

Cytoplasmic/nuclear localization of tuberin in different cell lines.

Tuberous sclerosis (TSC) is an autosomal dominantly inherited disease affecting 1 in 6000 individuals. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component being involved in a wide variety of different cellular processes. Tuberin has been demonstrated to be localized to both, the cytoplasm and the nucleus. The cytoplasmic/nuclear localization of tuberin is known to be regulated by the serine/threonine protein kinase Akt. Akt also regulates the cytoplasmic/nuclear localization of the cyclin-dependent kinase inhibitor p27. In this study the localization of these two Akt-regulated proteins was analysed in different cell lines.

Nuclear/cytoplasmic localization of Akt activity in the cell cycle.

The serine/threonine protein kinase Akt (also known as PKB) is a proto-oncogene and one of the most frequently hyperactivated kinases in human cancer. Its activation downstream of growth-factor-stimulated phosphatidylinositide-3'-OH kinase activity plays a role in the control of cell cycle, cell growth, apoptosis and cell energy metabolism. Akt phosphorylates some thousand downstream substrates, including typical cytoplasmic as well as nuclear proteins. Accordingly, it is not surprising that Akt activity can be found in both, the cytoplasm and the nucleus. Here we report the cell cycle regulation of nuclear and cytoplasmic Akt activity in mammalian cells. These data provide new insights into the regulation of Akt activity and have implications for future studies on the regulation of the wide variety of different nuclear and cytoplasmic Akt substrates.

Akt regulates nuclear/cytoplasmic localization of tuberin.

The autosomal dominantly inherited disease tuberous sclerosis (TSC) affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. TSC1, encoding hamartin, and TSC2, encoding tuberin are tumor suppressor genes responsible for TSC. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR)/p70S6K cascade. Phosphorylation of S939 and T1462 by Akt downregulates tuberin's potential to inhibit mTOR/p70S6K. Here, we show that this tuberin phosphorylation by Akt does not affect tuberin-mediated control of p27 protein amounts. This demonstrates that regulating p27 protein amounts and mTOR/p70S6K are separable functions of tuberin. Furthermore, we found that phosphorylation by Akt triggers upregulation of cytoplasmic and downregulation of nuclear tuberin. In cycling cells with high Akt activity, tuberin is predominantly localized to the cytoplasm. In arrested G0 cells with downregulated Akt activity, a significant proportion of tuberin is localized to the nucleus. Upon re-entry into the normal ongoing cell cycle, nuclear localization of tuberin is downregulated parallel to the activation of Akt. Recently, the mTOR/p70S6K cascade has been demonstrated to exist in both the cytoplasm and nucleus. We here also found that tuberin harbors the potential to regulate p70S6K activity in both the cytoplasm and nucleus. This description of functional tuberin in the cytoplasm and the nucleus together with our observation of Akt-controlled and cell cycle-regulated tuberin localization are of particular interest for a further understanding of tuberin's function as a gate keeper of the G0 cell status as well as of Akt's activity to control cell proliferation.

Tuberin negatively affects BCL-2's cell survival function.

Uncontrolled cell cycle progression and cell growth are key properties of tumor cells. The tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC) have been demonstrated to control both, cell cycle and cell size regulation. Hamartin, encoded by TSC1, and tuberin, encoded by TSC2, form a complex, of which tuberin is assumed to be the functional component. Loss of TSC genes function triggers hamartoma development in TSC patients. However, in vivo mostly tumor cell development is rapidly terminated via apoptosis. BCL-2, the founding member of the BCL-2 family of proteins, is well known for its anti-apoptotic properties. Here we show that pro-apoptotic actinomycin D cannot interfere with BCL-2's cell survival functions. However, we found tuberin to negatively regulate BCL-2's anti-apoptotic effects on low serum-induced apoptosis. These findings warrant further investigations to elucidate the molecular mechanism underlying tuberin's negative effects on cell survival.

Tuberin activates the proapoptotic molecule BAD.

TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.