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Melanoma brain colonization involves the emergence of a brain-adaptive phenotype.

Nygaard V, Prasmickaite L, Vasiliauskaite K, Clancy T, Hovig E - Oncoscience (2014)

Bottom Line: The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain.Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases.Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0310, Norway.

ABSTRACT
The brain offers a unique microenvironment that plays an important role in the establishment and progression of metastasis. However, the molecular determinants that promote development of melanoma brain metastases are largely unknown. Utilizing two species of immune-compromised animals, with in vivo cultivated metastatic tissues along with their corresponding host tissues in a metastasis model, we here identify molecular events associated with melanoma brain metastases. We find that the transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, reveal the opportunistic nature of melanoma in this biological context in rewiring the molecular framework of key molecular players with their associated biological processes. Specifically, we identify the existence of a neuron-like melanoma phenotype, which includes synaptic characteristics and a neurotransmission-like circuit involving glutamate. Regulation of gene transcription and neuron-like plasticity by Ca(2+)-dependent signaling appear to occur through glutamate receptor activation. The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain. Analysis of the host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells that permitted melanoma growth at the expense of the host organism. Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases. Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

No MeSH data available.


Related in: MedlinePlus

Glutamate receptor signaling and immunohistochemical validation of in vitro and in vivo(A) Glutamate concentration measured in medium after 72 hours of culture. Cells were plated in 96-well plates at 5x103 cells per well and 10 μl were collected (10% of media) (B-C) The AMPA antagonists SYM2206 and CFM-2 exert antiproliferative effects on both Melmet cell lines. Cells were exposed to vehicle control (DMSO) or 5-200 μM SYM2206 or CFM-2 for 72 hours, and viability was measured by means of the MTS assay. (D) The CaMKII inhibitor, KN93, decreased viability of both Melmet cell lines in the MTS assay. (E) (Upper panel) Representative H&E sections stained for the glutamate receptor, GRIA2 based on fixated MM5 cells from in vitro cultures and MM5 brain, lung and tibia metastases obtained from the experimental metastasis mouse model. The brain section showed stronger staining of GRIA2 with a nuclear localization. (Lower panel) The brain metastasis section from the patient from which the MM1 cell line was derived, also showed stronger nuclear staining of GRIA2 compared to the patient-matched subcutaneous lesion. See Supplementary Fig. S5C for additional MM1 stained sections.
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Figure 7: Glutamate receptor signaling and immunohistochemical validation of in vitro and in vivo(A) Glutamate concentration measured in medium after 72 hours of culture. Cells were plated in 96-well plates at 5x103 cells per well and 10 μl were collected (10% of media) (B-C) The AMPA antagonists SYM2206 and CFM-2 exert antiproliferative effects on both Melmet cell lines. Cells were exposed to vehicle control (DMSO) or 5-200 μM SYM2206 or CFM-2 for 72 hours, and viability was measured by means of the MTS assay. (D) The CaMKII inhibitor, KN93, decreased viability of both Melmet cell lines in the MTS assay. (E) (Upper panel) Representative H&E sections stained for the glutamate receptor, GRIA2 based on fixated MM5 cells from in vitro cultures and MM5 brain, lung and tibia metastases obtained from the experimental metastasis mouse model. The brain section showed stronger staining of GRIA2 with a nuclear localization. (Lower panel) The brain metastasis section from the patient from which the MM1 cell line was derived, also showed stronger nuclear staining of GRIA2 compared to the patient-matched subcutaneous lesion. See Supplementary Fig. S5C for additional MM1 stained sections.

Mentions: Based on the involvement of glutamate receptors in the brain-signature, we investigated the presence of functional glutamate signaling in MM1 and MM5 in vitro. We first established that MM1 raised the level of extracellular glutamate in the culture medium six times more than MM5 after 72 hours (Fig. 7A). GRIA2 is a subunit of the AMPA glutamate receptor family (GRIA1-4), and with real time PCR analyses, we detected low and differential expression of the AMPA subunits in MM1 and MM5 (Supporting Information Fig. S5A). Blockade of AMPA receptors by means of the antagonists CFM-2 and SYM2206 suppressed cell viability of both cell lines (Fig. 7B-C). Glutamate receptor activation allows the influx of Ca2+ and activation of downstream Ca2+-dependent effectors. Both cell lines showed sensitivity to the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN93 (Fig. 7D). To verify the induction of neuronal gene expression in our metastatic melanoma cell lines when grown in a brain microenvironment, we cultured MM1 in brain-conditioned medium in vitro. The expression of GRIA2, GRM4 and BSN increased in the presence of brain-conditioned medium relative to control medium (Supporting Information Fig. S5B). Immunohistochemical detection of GRIA2 was performed on metastatic sections from both the animal model and on patient material. In the brain, GRIA2 staining was detected solely in the nuclear compartment where the staining was stronger when compared to other metastatic sites (Fig. 7E and Supporting Information Fig. S5C). This was also observed in the metastatic tissue sections obtained from patient material from which the MM1 cell line was derived (Fig. 7E). Collectively, these data support a role for glutamate receptor signalling in cellular growth of MM1 and MM5 in vitro. The glutamate receptor signalling appears enhanced in the brain microenvironment, and thus represents a targeting opportunity.


Melanoma brain colonization involves the emergence of a brain-adaptive phenotype.

Nygaard V, Prasmickaite L, Vasiliauskaite K, Clancy T, Hovig E - Oncoscience (2014)

Glutamate receptor signaling and immunohistochemical validation of in vitro and in vivo(A) Glutamate concentration measured in medium after 72 hours of culture. Cells were plated in 96-well plates at 5x103 cells per well and 10 μl were collected (10% of media) (B-C) The AMPA antagonists SYM2206 and CFM-2 exert antiproliferative effects on both Melmet cell lines. Cells were exposed to vehicle control (DMSO) or 5-200 μM SYM2206 or CFM-2 for 72 hours, and viability was measured by means of the MTS assay. (D) The CaMKII inhibitor, KN93, decreased viability of both Melmet cell lines in the MTS assay. (E) (Upper panel) Representative H&E sections stained for the glutamate receptor, GRIA2 based on fixated MM5 cells from in vitro cultures and MM5 brain, lung and tibia metastases obtained from the experimental metastasis mouse model. The brain section showed stronger staining of GRIA2 with a nuclear localization. (Lower panel) The brain metastasis section from the patient from which the MM1 cell line was derived, also showed stronger nuclear staining of GRIA2 compared to the patient-matched subcutaneous lesion. See Supplementary Fig. S5C for additional MM1 stained sections.
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Related In: Results  -  Collection

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Figure 7: Glutamate receptor signaling and immunohistochemical validation of in vitro and in vivo(A) Glutamate concentration measured in medium after 72 hours of culture. Cells were plated in 96-well plates at 5x103 cells per well and 10 μl were collected (10% of media) (B-C) The AMPA antagonists SYM2206 and CFM-2 exert antiproliferative effects on both Melmet cell lines. Cells were exposed to vehicle control (DMSO) or 5-200 μM SYM2206 or CFM-2 for 72 hours, and viability was measured by means of the MTS assay. (D) The CaMKII inhibitor, KN93, decreased viability of both Melmet cell lines in the MTS assay. (E) (Upper panel) Representative H&E sections stained for the glutamate receptor, GRIA2 based on fixated MM5 cells from in vitro cultures and MM5 brain, lung and tibia metastases obtained from the experimental metastasis mouse model. The brain section showed stronger staining of GRIA2 with a nuclear localization. (Lower panel) The brain metastasis section from the patient from which the MM1 cell line was derived, also showed stronger nuclear staining of GRIA2 compared to the patient-matched subcutaneous lesion. See Supplementary Fig. S5C for additional MM1 stained sections.
Mentions: Based on the involvement of glutamate receptors in the brain-signature, we investigated the presence of functional glutamate signaling in MM1 and MM5 in vitro. We first established that MM1 raised the level of extracellular glutamate in the culture medium six times more than MM5 after 72 hours (Fig. 7A). GRIA2 is a subunit of the AMPA glutamate receptor family (GRIA1-4), and with real time PCR analyses, we detected low and differential expression of the AMPA subunits in MM1 and MM5 (Supporting Information Fig. S5A). Blockade of AMPA receptors by means of the antagonists CFM-2 and SYM2206 suppressed cell viability of both cell lines (Fig. 7B-C). Glutamate receptor activation allows the influx of Ca2+ and activation of downstream Ca2+-dependent effectors. Both cell lines showed sensitivity to the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN93 (Fig. 7D). To verify the induction of neuronal gene expression in our metastatic melanoma cell lines when grown in a brain microenvironment, we cultured MM1 in brain-conditioned medium in vitro. The expression of GRIA2, GRM4 and BSN increased in the presence of brain-conditioned medium relative to control medium (Supporting Information Fig. S5B). Immunohistochemical detection of GRIA2 was performed on metastatic sections from both the animal model and on patient material. In the brain, GRIA2 staining was detected solely in the nuclear compartment where the staining was stronger when compared to other metastatic sites (Fig. 7E and Supporting Information Fig. S5C). This was also observed in the metastatic tissue sections obtained from patient material from which the MM1 cell line was derived (Fig. 7E). Collectively, these data support a role for glutamate receptor signalling in cellular growth of MM1 and MM5 in vitro. The glutamate receptor signalling appears enhanced in the brain microenvironment, and thus represents a targeting opportunity.

Bottom Line: The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain.Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases.Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0310, Norway.

ABSTRACT
The brain offers a unique microenvironment that plays an important role in the establishment and progression of metastasis. However, the molecular determinants that promote development of melanoma brain metastases are largely unknown. Utilizing two species of immune-compromised animals, with in vivo cultivated metastatic tissues along with their corresponding host tissues in a metastasis model, we here identify molecular events associated with melanoma brain metastases. We find that the transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, reveal the opportunistic nature of melanoma in this biological context in rewiring the molecular framework of key molecular players with their associated biological processes. Specifically, we identify the existence of a neuron-like melanoma phenotype, which includes synaptic characteristics and a neurotransmission-like circuit involving glutamate. Regulation of gene transcription and neuron-like plasticity by Ca(2+)-dependent signaling appear to occur through glutamate receptor activation. The brain-adaptive phenotype was found as more prominent in the early metastatic growth phases compared to a later phase, emphasizing a temporal requirement of critical events in the successful colonization of the brain. Analysis of the host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells that permitted melanoma growth at the expense of the host organism. Combined experimental and computational approaches clearly highlighted genes and signaling pathways being shared with neurodegenerative diseases. Importantly, the identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

No MeSH data available.


Related in: MedlinePlus