Modulation of RAB5A early endosome trafficking in response to KRas mediated macropinocytic fluxes in pancreatic cancer cells

https://doi.org/10.1016/j.bbrc.2017.08.157Get rights and content

Highlights

  • Mutant KRAS promotes endosomal degradation in PDAC cell lines.

  • The endocytic machinery is not identically regulated in different PDACs.

  • KRAS silencing activates RAB5A early endosome trafficking in subtypes of PDAC.

  • RAB5A overexpression in turn most likely enlarges the early endosome compartment.

Abstract

KRAS is the key mutated gene in pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that KRas modulates endocytic uptake. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression in PDAC.

Surprisingly, PANC-1 cells lacking KRas exhibited significantly enlarged early and late endosomes containing internalized dextran and epidermal growth factor. Endosome enlargement was accompanied by reduced endosomal degradation. Both KRas silencing and lysosomal blockade caused an upregulation of the master regulator of early endosome biogenesis, RAB5A, which is likely responsible for the expansion of the early endosomal compartment, because simultaneous KRAS/RAB5A knockdown abolished endosome enlargement. In contrast, early endosome shrinkage was seen in MIA PaCa-2 cells despite RAB5A upregulation, indicating that distinct KRas-modulated responses operate in different metabolic subtypes of PDAC.

In conclusion, mutant KRAS promotes endosomal degradation in PDAC cell lines, which is impaired by KRAS silencing. Moreover, KRAS silencing activates RAB5A upregulation and drives PDAC subtype-dependent modulation of endosome trafficking.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Less than 5% of affected patients can expect long-term survival, even if the primary, localized tumor is curatively resected and patients undergo adjuvant chemotherapy [1]. Early lymph node metastasis and high resistance toward chemotherapy are essential reasons for the aggressive growth of this malignancy. More than 90% of the PDAC cell lines exhibit activating mutations of the KRAS gene, which is considered the signature mutation within a diverse mutational landscape of the tumors, including genetic and metabolic subtypes [2], [3]. KRas primarily localizes to the plasma membrane, but can also translocate to early endosomes following the endocytic pathway. Oncogenic KRAS thereby stimulates macropinocytosis and autophagy to ensure an extra nutrient supply, and to exploit metabolic needs and energy stores (such as during anticancer treatments) [4], [5], [6], [7]. Macropinocytosis, autophagy, and signaling events rely on endocytosis and recycling of endosome vesicles. Briefly, the machinery of vesicle degradation consists of the maturation of early endosomes, trafficking, their fusion with the lysosomal compartment, and the hydrolytic cleavage of their content.

One of the master regulators of this process is RAB5, which is a critical determinant of endosomal cargo sorting to degradation or recycling routes, and is essential for macropinosome formation [8], [9], [10]. In several types of cancer, RAB5 has been associated with increased tumor invasion and dissemination [11], [12]. To date, no such data exist that support the involvement of RAB5 in PDAC progression. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression and activity in PDAC.

Section snippets

Cell lines

The experimental protocol was approved by the local ethics committee of the Technische Universität Dresden (decision number EK 496122016). The human pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were purchased from ATCC (LGC Standards, Wesel, Germany) and regularly tested for contamination. PANC-1 and BxPC-3 were grown in an RPMI-1640 medium, supplemented with 10% fetal calf serum (FCS). For MIA PaCa-2 cells, Dulbecco's modified Eagle medium, including 10% FCS and 2.5% horse

KRas knockdown leads to enlarged early endosomes and a blockade of fluid phase endocytic flux

The PDAC cell lines, PANC-1, MIA PaCa-2 and BxPC-3, were all derived from primary tumors, and exhibited different expression levels of KRAS transcripts. The WT KRAS-expressing BxPC-3 cells had lower KRas mRNA expression than the two cell lines with activating mutations of KRAS. The latter two are classified into different metabolic subtypes: the glycolytic (MIA PaCa-2) and the lipogenic (PANC-1) subtypes [3]. The PANC-1 cell line with heterozygously mutated KRAS had the highest KRas expression (

Discussion

There is increasing evidence that the endocytic/lysosomal compartment significantly determines tumor progression of PDAC and other solid malignancies [12], [16]. The signature mutation KRAS contributes to the aberrant PDAC metabolism by promoting glucose and glutamine uptake, and by the stimulating of autophagy [7], [17], [18]. However, the metabolic profiling revealed a significant PDAC heterogeneity and enabled characterization of three metabolic subtypes, which exhibit different metabolic

Acknowledgments

The authors would like to thank Silke Tulok (Core Facility Cellular Imaging, MTZ, TU Dresden, Germany) for her excellent support and advice. This work was supported by a German Research Foundation (DFG) research grant to T.W. (WE 4816/1–2); a German Cancer Aid grant to C.T. and T.W.; the Associazione Italiana per la Ricerca sul Cancro (AIRC #10168 and #18621); Worldwide Cancer Research (AICR- 14-0335); and the European Research Council (Advanced-ERC-#268836) to G.S.

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