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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

Class I PI3K signaling drives constitutive RAB7-regulated macropinocytosis in melanoma cellsA.Ki,app values (in nM) of the indicated Class I PI3K inhibitors (see Methods for additional detail). B. Western blot analysis of SK-Mel-103 melanoma cells treated for the indicated times with Class I PI3K inhibitor GDC-0941 at the indicated concentrations, blotted for total and phosphorylated (p) AKT (Ser473). Vinculin is included as loading control. C. Representative bright field micrographs of shControl or shRAB7 SK-Mel-103 cells treated with DMSO (left) 0.5μM GDC-0941 (middle) or 0.5μM ETP-46992 (right) for 7h. The corresponding quantification of the impact of these treatments on cytosolic vacuolization is shown in D. E. Bright field micrographs showing the reversion of cytosolic vacuolization of SK-Mel-103 expressing RAB7 shRNAs by treatment with the indicated Class I PI3K inhibitors for 48h. F. Bright field, fluorescence and merged micrographs of the basal 8h-uptake of 70 kD Rhodamine(Rhd)-Dextran by SK-Mel-103 melanoma cells incubated in the absence or presence of the indicated Class I PI3K inhibitors for 48h.
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Figure 2: Class I PI3K signaling drives constitutive RAB7-regulated macropinocytosis in melanoma cellsA.Ki,app values (in nM) of the indicated Class I PI3K inhibitors (see Methods for additional detail). B. Western blot analysis of SK-Mel-103 melanoma cells treated for the indicated times with Class I PI3K inhibitor GDC-0941 at the indicated concentrations, blotted for total and phosphorylated (p) AKT (Ser473). Vinculin is included as loading control. C. Representative bright field micrographs of shControl or shRAB7 SK-Mel-103 cells treated with DMSO (left) 0.5μM GDC-0941 (middle) or 0.5μM ETP-46992 (right) for 7h. The corresponding quantification of the impact of these treatments on cytosolic vacuolization is shown in D. E. Bright field micrographs showing the reversion of cytosolic vacuolization of SK-Mel-103 expressing RAB7 shRNAs by treatment with the indicated Class I PI3K inhibitors for 48h. F. Bright field, fluorescence and merged micrographs of the basal 8h-uptake of 70 kD Rhodamine(Rhd)-Dextran by SK-Mel-103 melanoma cells incubated in the absence or presence of the indicated Class I PI3K inhibitors for 48h.

Mentions: Although LY294002 has been broadly used as a Class I PI3K inhibitor, this compound can also target other signalling cascades such as Class III PI3K [39]. Therefore, further analyses were performed with additional inhibitors targeting Class I PI3K more specifically. Given the complexity of Class I PI3K, with a catalytic p110 subunit constituted by one of four possible isoforms (α, β, δ and γ), in a heterodimeric complex with a regulatory subunit with also multiple variants [38], we opted for GDC-0941 [39], a well-known pan-p110 inhibitor (see pharmacological features of this compound in Figure 2A). Dose-response and kinetic analyses were performed in SK-Mel-103 to identify minimal effective concentrations for an efficient blockade of PI3K signalling, as defined by the abrogation of AKT phosphorylation in residue Ser473 (Figure 2B). As shown in Figure 2C, GDC-0941 reverted very efficiently the vacuolization induced in melanoma cells by RAB7 depletion (see the corresponding quantifications in Figure 2D). To independently validate these results, RAB7 shRNA-expressing melanoma cells were treated with ETP-46992 [40], a structurally different Class I pan PI3K blocker with an even more selective inhibitor profile (i.e. with reduced affinity for other kinases such as mTOR; see Figure 2A). ETP-46992 also resolved the aberrant vacuolization induced by RAB7 shRNA (Figure 2C, 2D). Importantly, ETP-38, a derivative of ETP-46992 (Figure 2A) that selectively binds and inactivates p110α and p110δ [41], also effectively rescued shRAB7-induced vacuolization (Figures 2E, 2D) and impaired constitutive macropinocytosis (Figure 2F), further narrowing down the p110 subunits that deregulate vesicular trafficking in a manner that is sensitive to RAB7 inhibition. Therefore, these data point to p110α and p110δ Class I PI3K as key activators of RAB7-dependent macroendocytic trafficking in melanoma cells.


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)

Class I PI3K signaling drives constitutive RAB7-regulated macropinocytosis in melanoma cellsA.Ki,app values (in nM) of the indicated Class I PI3K inhibitors (see Methods for additional detail). B. Western blot analysis of SK-Mel-103 melanoma cells treated for the indicated times with Class I PI3K inhibitor GDC-0941 at the indicated concentrations, blotted for total and phosphorylated (p) AKT (Ser473). Vinculin is included as loading control. C. Representative bright field micrographs of shControl or shRAB7 SK-Mel-103 cells treated with DMSO (left) 0.5μM GDC-0941 (middle) or 0.5μM ETP-46992 (right) for 7h. The corresponding quantification of the impact of these treatments on cytosolic vacuolization is shown in D. E. Bright field micrographs showing the reversion of cytosolic vacuolization of SK-Mel-103 expressing RAB7 shRNAs by treatment with the indicated Class I PI3K inhibitors for 48h. F. Bright field, fluorescence and merged micrographs of the basal 8h-uptake of 70 kD Rhodamine(Rhd)-Dextran by SK-Mel-103 melanoma cells incubated in the absence or presence of the indicated Class I PI3K inhibitors for 48h.
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Figure 2: Class I PI3K signaling drives constitutive RAB7-regulated macropinocytosis in melanoma cellsA.Ki,app values (in nM) of the indicated Class I PI3K inhibitors (see Methods for additional detail). B. Western blot analysis of SK-Mel-103 melanoma cells treated for the indicated times with Class I PI3K inhibitor GDC-0941 at the indicated concentrations, blotted for total and phosphorylated (p) AKT (Ser473). Vinculin is included as loading control. C. Representative bright field micrographs of shControl or shRAB7 SK-Mel-103 cells treated with DMSO (left) 0.5μM GDC-0941 (middle) or 0.5μM ETP-46992 (right) for 7h. The corresponding quantification of the impact of these treatments on cytosolic vacuolization is shown in D. E. Bright field micrographs showing the reversion of cytosolic vacuolization of SK-Mel-103 expressing RAB7 shRNAs by treatment with the indicated Class I PI3K inhibitors for 48h. F. Bright field, fluorescence and merged micrographs of the basal 8h-uptake of 70 kD Rhodamine(Rhd)-Dextran by SK-Mel-103 melanoma cells incubated in the absence or presence of the indicated Class I PI3K inhibitors for 48h.
Mentions: Although LY294002 has been broadly used as a Class I PI3K inhibitor, this compound can also target other signalling cascades such as Class III PI3K [39]. Therefore, further analyses were performed with additional inhibitors targeting Class I PI3K more specifically. Given the complexity of Class I PI3K, with a catalytic p110 subunit constituted by one of four possible isoforms (α, β, δ and γ), in a heterodimeric complex with a regulatory subunit with also multiple variants [38], we opted for GDC-0941 [39], a well-known pan-p110 inhibitor (see pharmacological features of this compound in Figure 2A). Dose-response and kinetic analyses were performed in SK-Mel-103 to identify minimal effective concentrations for an efficient blockade of PI3K signalling, as defined by the abrogation of AKT phosphorylation in residue Ser473 (Figure 2B). As shown in Figure 2C, GDC-0941 reverted very efficiently the vacuolization induced in melanoma cells by RAB7 depletion (see the corresponding quantifications in Figure 2D). To independently validate these results, RAB7 shRNA-expressing melanoma cells were treated with ETP-46992 [40], a structurally different Class I pan PI3K blocker with an even more selective inhibitor profile (i.e. with reduced affinity for other kinases such as mTOR; see Figure 2A). ETP-46992 also resolved the aberrant vacuolization induced by RAB7 shRNA (Figure 2C, 2D). Importantly, ETP-38, a derivative of ETP-46992 (Figure 2A) that selectively binds and inactivates p110α and p110δ [41], also effectively rescued shRAB7-induced vacuolization (Figures 2E, 2D) and impaired constitutive macropinocytosis (Figure 2F), further narrowing down the p110 subunits that deregulate vesicular trafficking in a manner that is sensitive to RAB7 inhibition. Therefore, these data point to p110α and p110δ Class I PI3K as key activators of RAB7-dependent macroendocytic trafficking in melanoma cells.

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