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RAB7 counteracts PI3K-driven macropinocytosis activated at early stages of melanoma development.

Alonso-Curbelo D, Osterloh L, Cañón E, Calvo TG, Martínez-Herranz R, Karras P, Martínez S, Riveiro-Falkenbach E, Romero PO, Rodríguez-Peralto JL, Pastor J, Soengas MS - Oncotarget (2015)

Bottom Line: Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined.Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells.In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes.

View Article: PubMed Central - PubMed

Affiliation: Melanoma Laboratory, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

ABSTRACT
Derailed endolysosomal trafficking is emerging as a widespread feature of aggressive neoplasms. However, the oncogenic signals that alter membrane homeostasis and their specific contribution to cancer progression remain unclear. Understanding the upstream drivers and downstream regulators of aberrant vesicular trafficking is distinctly important in melanoma. This disease is notorious for its inter- and intra-tumoral heterogeneity. Nevertheless, melanomas uniformly overexpress a cluster of endolysosomal genes, being particularly addicted to the membrane traffic regulator RAB7. Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined. Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells. This strategy revealed Class I PI3K as the key trigger of a hyperactive influx of macropinosomes that melanoma cells counteract via RAB7-mediated lysosomal degradation. In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes. Together, these results provide new insight into the regulators and modes of action of RAB7, broadening the impact of endosomal fitness on melanoma development.

No MeSH data available.


Related in: MedlinePlus

Recruitment of RAB7 to PI3K-driven macropinosomes upon oncogene activation in human melanocytesA. Representative IF staining for RAB7 (green) in primary human melanocytes expressing empty vector or oncogenic HRASG12V. Nuclei are counterstained with DAPI. B. Transmission electron microscopy image of primary human melanocytes expressing empty vector or HRASG12V. Note the presence of large single membrane macropinosomes in HRASG12V-expressing melanocytes, absent in the empty vector-expressing counterparts. C. Representative fluorescence, bright field and merged micrographs showing the activation of macropinocytic uptake (visualized by 70kD Rhodamine(Rhd)-Dextran uptake) in primary human melanocytes expressing oncogenic HRASG12V. D. Representative bright field micrographs showing senescence-associated β-Galactosidase (β-Gal) stainings of melanocytes transduced with empty HRASG12V-encoding vectors and treated with 10μM LY294002, 10 μM U0126 or vehicle control. Inhibitors were added one day post-transduction and were refreshed every 24h. Pictures were taken at day 6 post-transduction. E. Pooled quantification of vacuolized and β-Gal-positive cells from two independent experiments in vector- or HRASG12V- expressing melanocytes treated as indicated. Unless otherwise indicated, scale bars correspond to 10 μm.
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Figure 3: Recruitment of RAB7 to PI3K-driven macropinosomes upon oncogene activation in human melanocytesA. Representative IF staining for RAB7 (green) in primary human melanocytes expressing empty vector or oncogenic HRASG12V. Nuclei are counterstained with DAPI. B. Transmission electron microscopy image of primary human melanocytes expressing empty vector or HRASG12V. Note the presence of large single membrane macropinosomes in HRASG12V-expressing melanocytes, absent in the empty vector-expressing counterparts. C. Representative fluorescence, bright field and merged micrographs showing the activation of macropinocytic uptake (visualized by 70kD Rhodamine(Rhd)-Dextran uptake) in primary human melanocytes expressing oncogenic HRASG12V. D. Representative bright field micrographs showing senescence-associated β-Galactosidase (β-Gal) stainings of melanocytes transduced with empty HRASG12V-encoding vectors and treated with 10μM LY294002, 10 μM U0126 or vehicle control. Inhibitors were added one day post-transduction and were refreshed every 24h. Pictures were taken at day 6 post-transduction. E. Pooled quantification of vacuolized and β-Gal-positive cells from two independent experiments in vector- or HRASG12V- expressing melanocytes treated as indicated. Unless otherwise indicated, scale bars correspond to 10 μm.

Mentions: Immunofluorescence analyses of endogenous RAB proteins and endolysosomal markers (i.e. LAMP1) revealed an overt deregulation of the endolysosomal pathway in HRASG12V-expressing melanocytes, characterized by recruitment of RAB7 to the enlarged vacuolar structures induced by this oncogene (Figures 3A, S2A and results not shown). These large RAB7-positive vacuoles could originate from the plasma membrane, or from intracellular organelles such as the endoplasmic reticulum, the Golgi, and/or various recycling endomembranes [10, 45, 46]. In addition, RAB7 controls the fate of double-membrane autophagosomes [21], of relevance in oncogene-induced senescence in embryonic fibroblasts [47]. Addressing these different sources is important as a variety of key pathways involving and cooperating with RAS-dependent effectors can signal in a differential manner depending on their membrane-associated localization [48]. To this end, HRASG12V transduced melanocytes were processed for imaging by electron microscopy for a direct analysis of the ultrastructural cellular changes driven by this oncogene. This technique revealed that over 80% of vacuoles in HRASG12V-transduced melanocytes in fact corresponded to single-membrane vesicles (Figure 3B). The size of these vesicles, from 0.2 to over 2 μm (Figures 3A, 3B), their ability to uptake large solutes (70kDa-Dextran) from the extracellular space (Figure 3C), as well as their formation from actin-rich membrane ruffling (Figure S2B), support plasma-membrane driven macropinocytosis as the source of oncogene-driven vesicles that recruit RAB7 downstream of HRASG12V. As both these oncogenic signals and macropinocytosis are absent in normal melanocytes, these results provide further mechanistic evidence as to why melanomas are significantly more dependent on RAB7 than their normal cellular counterparts [13].


RAB7 counteracts PI3K-driven macropinocytosis activated at early stages of melanoma development.

Alonso-Curbelo D, Osterloh L, Cañón E, Calvo TG, Martínez-Herranz R, Karras P, Martínez S, Riveiro-Falkenbach E, Romero PO, Rodríguez-Peralto JL, Pastor J, Soengas MS - Oncotarget (2015)

Recruitment of RAB7 to PI3K-driven macropinosomes upon oncogene activation in human melanocytesA. Representative IF staining for RAB7 (green) in primary human melanocytes expressing empty vector or oncogenic HRASG12V. Nuclei are counterstained with DAPI. B. Transmission electron microscopy image of primary human melanocytes expressing empty vector or HRASG12V. Note the presence of large single membrane macropinosomes in HRASG12V-expressing melanocytes, absent in the empty vector-expressing counterparts. C. Representative fluorescence, bright field and merged micrographs showing the activation of macropinocytic uptake (visualized by 70kD Rhodamine(Rhd)-Dextran uptake) in primary human melanocytes expressing oncogenic HRASG12V. D. Representative bright field micrographs showing senescence-associated β-Galactosidase (β-Gal) stainings of melanocytes transduced with empty HRASG12V-encoding vectors and treated with 10μM LY294002, 10 μM U0126 or vehicle control. Inhibitors were added one day post-transduction and were refreshed every 24h. Pictures were taken at day 6 post-transduction. E. Pooled quantification of vacuolized and β-Gal-positive cells from two independent experiments in vector- or HRASG12V- expressing melanocytes treated as indicated. Unless otherwise indicated, scale bars correspond to 10 μm.
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Figure 3: Recruitment of RAB7 to PI3K-driven macropinosomes upon oncogene activation in human melanocytesA. Representative IF staining for RAB7 (green) in primary human melanocytes expressing empty vector or oncogenic HRASG12V. Nuclei are counterstained with DAPI. B. Transmission electron microscopy image of primary human melanocytes expressing empty vector or HRASG12V. Note the presence of large single membrane macropinosomes in HRASG12V-expressing melanocytes, absent in the empty vector-expressing counterparts. C. Representative fluorescence, bright field and merged micrographs showing the activation of macropinocytic uptake (visualized by 70kD Rhodamine(Rhd)-Dextran uptake) in primary human melanocytes expressing oncogenic HRASG12V. D. Representative bright field micrographs showing senescence-associated β-Galactosidase (β-Gal) stainings of melanocytes transduced with empty HRASG12V-encoding vectors and treated with 10μM LY294002, 10 μM U0126 or vehicle control. Inhibitors were added one day post-transduction and were refreshed every 24h. Pictures were taken at day 6 post-transduction. E. Pooled quantification of vacuolized and β-Gal-positive cells from two independent experiments in vector- or HRASG12V- expressing melanocytes treated as indicated. Unless otherwise indicated, scale bars correspond to 10 μm.
Mentions: Immunofluorescence analyses of endogenous RAB proteins and endolysosomal markers (i.e. LAMP1) revealed an overt deregulation of the endolysosomal pathway in HRASG12V-expressing melanocytes, characterized by recruitment of RAB7 to the enlarged vacuolar structures induced by this oncogene (Figures 3A, S2A and results not shown). These large RAB7-positive vacuoles could originate from the plasma membrane, or from intracellular organelles such as the endoplasmic reticulum, the Golgi, and/or various recycling endomembranes [10, 45, 46]. In addition, RAB7 controls the fate of double-membrane autophagosomes [21], of relevance in oncogene-induced senescence in embryonic fibroblasts [47]. Addressing these different sources is important as a variety of key pathways involving and cooperating with RAS-dependent effectors can signal in a differential manner depending on their membrane-associated localization [48]. To this end, HRASG12V transduced melanocytes were processed for imaging by electron microscopy for a direct analysis of the ultrastructural cellular changes driven by this oncogene. This technique revealed that over 80% of vacuoles in HRASG12V-transduced melanocytes in fact corresponded to single-membrane vesicles (Figure 3B). The size of these vesicles, from 0.2 to over 2 μm (Figures 3A, 3B), their ability to uptake large solutes (70kDa-Dextran) from the extracellular space (Figure 3C), as well as their formation from actin-rich membrane ruffling (Figure S2B), support plasma-membrane driven macropinocytosis as the source of oncogene-driven vesicles that recruit RAB7 downstream of HRASG12V. As both these oncogenic signals and macropinocytosis are absent in normal melanocytes, these results provide further mechanistic evidence as to why melanomas are significantly more dependent on RAB7 than their normal cellular counterparts [13].

Bottom Line: Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined.Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells.In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes.

View Article: PubMed Central - PubMed

Affiliation: Melanoma Laboratory, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

ABSTRACT
Derailed endolysosomal trafficking is emerging as a widespread feature of aggressive neoplasms. However, the oncogenic signals that alter membrane homeostasis and their specific contribution to cancer progression remain unclear. Understanding the upstream drivers and downstream regulators of aberrant vesicular trafficking is distinctly important in melanoma. This disease is notorious for its inter- and intra-tumoral heterogeneity. Nevertheless, melanomas uniformly overexpress a cluster of endolysosomal genes, being particularly addicted to the membrane traffic regulator RAB7. Still, the underlying mechanisms and temporal determinants of this dependency have yet to be defined. Here we addressed these questions by combining electron microscopy, real time imaging and mechanistic analyses of vesicular trafficking in normal and malignant human melanocytic cells. This strategy revealed Class I PI3K as the key trigger of a hyperactive influx of macropinosomes that melanoma cells counteract via RAB7-mediated lysosomal degradation. In addition, gain- and loss-of-function in vitro studies followed by histopathological validation in clinical biopsies and genetically-engineered mouse models, traced back the requirement of RAB7 to the suppression of premature cellular senescence traits elicited in melanocytes by PI3K-inducing oncogenes. Together, these results provide new insight into the regulators and modes of action of RAB7, broadening the impact of endosomal fitness on melanoma development.

No MeSH data available.


Related in: MedlinePlus