Depletion of pyruvate kinase (PK) activity causes glycolytic intermediate imbalances and reveals a PK-TXNIP regulatory axis.

OBJECTIVE: Cancer cells convert more glucose into lactate than healthy cells, what contributes to their growth advantage. Pyruvate kinase (PK) is a key rate limiting enzyme in this process, what makes it a promising potential therapeutic target. However, currently it is still unclear what consequences the inhibition of PK has on cellular processes. Here, we systematically investigate the consequences of PK depletion for gene expression, histone modifications and metabolism. METHODS: Epigenetic, transcriptional and metabolic targets were analysed in different cellular and animal models with stable knockdown or knockout of PK. RESULTS: Depleting PK activity reduces the glycolytic flux and causes accumulation of glucose-6-phosphate (G6P). Such metabolic perturbation results in stimulation of the activity of a heterodimeric pair of transcription factors MondoA and MLX but not in a major reprogramming of the global H3K9ac and H3K4me3 histone modification landscape. The MondoA:MLX heterodimer upregulates expression of thioredoxin-interacting protein (TXNIP) - a tumour suppressor with multifaceted anticancer activity. This effect of TXNIP upregulation extends beyond immortalised cancer cell lines and is applicable to multiple cellular and animal models. CONCLUSIONS: Our work shows that actions of often pro-tumorigenic PK and anti-tumorigenic TXNIP are tightly linked via a glycolytic intermediate. We suggest that PK depletion stimulates the activity of MondoA:MLX transcription factor heterodimers and subsequently, increases cellular TXNIP levels. TXNIP-mediated inhibition of thioredoxin (TXN) can reduce the ability of cells to scavenge reactive oxygen species (ROS) leading to the oxidative damage of cellular structures including DNA. These findings highlight an important regulatory axis affecting tumour suppression mechanisms and provide an attractive opportunity for combination cancer therapies targeting glycolytic activity and ROS-generating pathways.

Isoform-specific and ubiquitination dependent recruitment of Tet1 to replicating heterochromatin modulates methylcytosine oxidation.

Oxidation of the epigenetic DNA mark 5-methylcytosine by Tet dioxygenases is an established route to diversify the epigenetic information, modulate gene expression and overall cellular (patho-)physiology. Here, we demonstrate that Tet1 and its short isoform Tet1s exhibit distinct nuclear localization during DNA replication resulting in aberrant cytosine modification levels in human and mouse cells. We show that Tet1 is tethered away from heterochromatin via its zinc finger domain, which is missing in Tet1s allowing its targeting to these regions. We find that Tet1s interacts with and is ubiquitinated by CRL4(VprBP). The ubiquitinated Tet1s is then recognized by Uhrf1 and recruited to late replicating heterochromatin. This leads to spreading of 5-methylcytosine oxidation to heterochromatin regions, LINE 1 activation and chromatin decondensation. In summary, we elucidate a dual regulation mechanism of Tet1, contributing to the understanding of how epigenetic information can be diversified by spatio-temporal directed Tet1 catalytic activity.

Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice.

UNLABELLED: Due to its ability to inhibit prometastatic matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP)-1 has been thought to suppress tumor metastasis. However, elevated systemic levels of TIMP-1 correlate with poor prognosis in cancer patients, suggesting a metastasis-stimulating role of TIMP-1. In colorectal cancer patients, tumor as well as plasma TIMP-1 levels were correlated with synchronous liver metastasis or distant metastasis-associated disease relapse. In mice, high systemic TIMP-1 levels increased the liver susceptibility towards metastasis by triggering the formation of a premetastatic niche. This promoted hepatic metastasis independent of origin or intrinsic metastatic potential of tumor cells. High systemic TIMP-1 led to increased hepatic SDF-1 levels, which in turn promoted recruitment of neutrophils to the liver. Both inhibition of SDF-1-mediated neutrophil recruitment and systemic depletion of neutrophils reduced TIMP-1-induced increased liver susceptibility towards metastasis. This indicates a crucial functional role of neutrophils in the TIMP-1-induced premetastatic niche. CONCLUSION: Our results identify TIMP-1 as an essential promoter of hepatic premetastatic niche formation.

The MET Oncogene as a Therapeutical Target in Cancer Invasive Growth.

The MET proto-oncogene, encoding the tyrosine kinase receptor for Hepatocyte Growth Factor (HGF) regulates invasive growth, a genetic program that associates control of cell proliferation with invasion of the extracellular matrix and protection from apoptosis. Physiologically, invasive growth takes place during embryonic development, and, in post-natal life, in wound healing and regeneration of several tissues. The MET oncogene is overexpressed and/or genetically mutated in many tumors, thereby sustaining pathological invasive growth, a prerequisite for metastasis. MET is the subject of intense research as a target for small molecule kinase inhibitors and, together with its ligand HGF, for inhibitory antibodies. The tight interplay of MET with the protease network has unveiled mechanisms to be exploited to achieve effective inhibition of invasive growth.

On the Pro-Metastatic Stress Response to Cancer Therapies: Evidence for a Positive Co-Operation between TIMP-1, HIF-1α, and miR-210.

In contrast to expectations in the past that tumor starvation or unselective inhibition of proteolytic activity would cure cancer, there is accumulating evidence that microenvironmental stress, such as hypoxia or broad-spectrum inhibition of metalloproteinases can promote metastasis. In fact, malignant tumor cells, due to their genetic and epigenetic instability, are predisposed to react to stress by adaptation and, if the stress persists, by escape and formation of metastasis. Recent recognition of the concepts of dynamic evolution as well as population and systems biology is extremely helpful to understand the disappointments of clinical trials with new drugs and may lead to paradigm-shifts in therapy strategies. This must be complemented by an increased understanding of molecular mechanism involved in stress response. Here, we review new roles of Hypoxia-inducible factor-1 (HIF-1), one transcription factor regulating stress response-related gene expression: HIF-1 is crucial for invasion and metastasis, independent from its pro-survival function. In addition, HIF-1 mediates pro-metastatic microenvironmental changes of the proteolytic balance as triggered by high systemic levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), typical for many aggressive cancers, and regulates the metabolic switch to glycolysis, notably via activation of the microRNA miR-210. There is preliminary evidence that TIMP-1 also induces miR-210. Such positive-regulatory co-operation of HIF-1α, miR-210, and TIMP-1, all described to correlate with bad prognosis of cancer patients, opens new perspectives of gaining insight into molecular mechanisms of metastasis-inducing evasion of tumor cells from stress.

Tumor cell-derived Timp-1 is necessary for maintaining metastasis-promoting Met-signaling via inhibition of Adam-10.

In many different tumor entities, increased expression of tissue inhibitor of metalloproteinases-1 (Timp-1) is associated with poor prognosis. We previously reported in mouse models that elevated systemic levels of Timp-1 induce a gene expression signature in the liver microenvironment increasing the susceptibility of this organ to tumor cells. This host effect was dependent on increased activity of the hepatocyte growth factor (Hgf)/hepatocyte growth factor receptor (Met) signaling pathway. In a recent study we showed that Met signaling is regulated by Timp-1 as it inhibits the Met sheddase A disintegrin and metalloproteinase-10 (Adam-10). The aim of the present study was to elucidate whether the metastatic potential of tumor cells benefits from autocrine Timp-1 as well and involves Adam-10 and Met signaling. In a syngeneic murine model of experimental liver metastasis Timp-1 expression and Met signaling were localized within metastatic colonies and expressed by tumor cells. Knock down of tumor cell Timp-1 suppressed Met signaling in metastases and inhibited metastasis formation and tumor cell-scattering in the liver. In vitro, knock down of tumor cell Timp-1 prevented Hgf-induced Met phosphorylation. Consequently, knock down of Met sheddase Adam-10 triggered auto-phosphorylation and responsiveness to Hgf. Accordingly, Adam-10 knock down increased Met phosphorylation in metastatic foci and induced tumor cell scattering into the surrounding liver parenchyma. In conclusion, these findings show that tumor cell-derived Timp-1 acts as a positive regulator of the metastatic potential and support the concept that proteases and their natural inhibitors, as members of the protease web, are major players of signaling during normal homeostasis and disease.

Full-length L1CAM and not its Δ2Δ27 splice variant promotes metastasis through induction of gelatinase expression.

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.

Tissue inhibitor of metalloproteinases-1-induced scattered liver metastasis is mediated by hypoxia-inducible factor-1α.

The "protease web", representing the network of proteases, their inhibitors, and effector molecules, arises as a pivotal determinant of tissue homeostasis. Imbalances of this network, for instance caused by elevated host levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), have been shown to increase the susceptibility of target organs to scattered metastasis by inducing the hepatocyte growth factor (HGF) pathway. Increased expression of the hypoxia-inducible factor-1α-subunit (HIF-1α) is also associated with tumour progression and is also known to induce HGF-signaling via up-regulation of the HGF-receptor Met, namely under canonical stress conditions like lack of oxygen. Here, we aimed to identify a possible metastasis-promoting connection between TIMP-1, HIF-1α, and HGF-signaling. We found that HIF-1α and HIF-1-signaling were increased during liver metastasis of L-CI.5s T-lymphoma cells in TIMP-1 overexpressing syngeneic DBA/2 mice. In vitro, exposure of L-CI.5s cells to recombinant TIMP-1 revealed that TIMP-1 itself was able to induce HIF-1α and HIF-1-signaling. Knock-down of HIF-1α identified tumour cell-derived HIF-1α as mediator of this TIMP-1-induced invasiveness in vitro. In vivo, HIF-1α knock-down significantly impaired Met expression as well as Met phosphorylation and inhibited scattered liver metastasis. Furthermore, HGF-dependent TIMP-1-promoted Met phosphorylation and HGF-dependent TIMP-1-induced invasiveness in vitro was mediated by HIF-1α. We conclude that elevated levels of TIMP-1 in the microenvironment of tumour cells can promote metastasis by inducing HIF-1α-dependent HGF-signaling. This connection between a protease inhibitor (TIMP-1) and a classically stress-related factor (HIF-1α) is a so far undiscovered impact of the "protease web" on tissue homeostasis with important implications for metastasis.

Identification of a survival-independent metastasis-enhancing role of hypoxia-inducible factor-1alpha with a hypoxia-tolerant tumor cell line.

During tumor progression, malignant cells must repeatedly survive microenvironmental stress. Hypoxia-inducible factor-1 (HIF-1) signaling has emerged as one major pathway allowing cellular adaptation to stress. Recent findings led to the hypothesis that HIF-1alpha may enhance the metastatic potential of tumor cells by a survival-independent mechanism. So far it has not been shown that HIF-1alpha also directly regulates invasive processes during metastasis in addition to conferring a survival advantage to metastasizing tumor cells. In a hypoxia-tolerant tumor cell line (L-CI.5s), which did not rely on HIF-1 signaling for viability in vitro and in vivo, knockdown of Hif-1alpha reduced invasiveness of the tumor cells in vitro as well as extravasation and secondary infiltration in vivo. Liver metastases associated induction of proinvasive receptor tyrosine kinase Met phosphorylation as well as gelatinolytic activity were Hif-1alpha-dependent. Indeed, promoter activity of the matrix metalloproteinase-9 (mmp-9) was shown to be Hif-1alpha-dependent. This study uncovers a new survival-independent biological function of HIF-1alpha contributing to the efficacy of metastases formation.

A disintegrin and metalloproteinase-10 (ADAM-10) mediates DN30 antibody-induced shedding of the met surface receptor.

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown. Here, we report that A Disintegrin And Metalloproteinase (ADAM)-10, but not ADAM-17, is required for DN30-induced Met shedding. Knockdown of ADAM-10 in different tumor cell lines or abrogation of its proteolytic activity by natural or synthetic inhibitors abolished Met down-regulation on the cell surface as well as reduction of Met activation. Moreover, hepatocyte growth factor-induced tumor cell migration and invasion were impaired upon ADAM-10 knockdown. Thus, the therapeutic effect of DN30 involves ADAM-10-dependent Met shedding, linking for the first time a specific metalloprotease to target therapy against a receptor tyrosine kinase.