Faculty Bibliography

Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents. Macropinocytosis is upregulated in oncogene-expressing cells and, recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past because of the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cells both in culture and growing in vivo as tumor xenografts. By using this approach, the 'macropinocytic index' of a particular cell line or subcutaneous tumor can be ascertained within 1-2 d. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft or allograft mouse models.

Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling.

RAS is the most frequently mutated oncogene in human cancers. Despite decades of effort, anti-RAS therapies have remained elusive. Isoprenylcysteine carboxylmethyltransferase (ICMT) methylates RAS and other CaaX-containing proteins, but its potential as a target for cancer therapy has not been fully evaluated. We crossed a Pdx1-Cre;LSL-KrasG12D mouse, which is a model of pancreatic ductal adenocarcinoma (PDA), with a mouse harboring a floxed allele of Icmt. Surprisingly, we found that ICMT deficiency dramatically accelerated the development and progression of neoplasia. ICMT-deficient pancreatic ductal epithelial cells had a slight growth advantage and were resistant to premature senescence by a mechanism that involved suppression of cyclin-dependent kinase inhibitor 2A (p16INK4A) expression. ICMT deficiency precisely phenocopied Notch1 deficiency in the Pdx1-Cre;LSL-KrasG12D model by exacerbating pancreatic intraepithelial neoplasias, promoting facial papillomas, and derepressing Wnt signaling. Silencing ICMT in human osteosarcoma cells decreased Notch1 signaling in response to stimulation with cell-surface ligands. Additionally, targeted silencing of Ste14, the Drosophila homolog of Icmt, resulted in defects in wing development, consistent with Notch loss of function. Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling.

BACKGROUND: The BRAF V600E (BRAF+) mutation activates the mitogen-activated protein kinase (MAPK/ERK) pathway and may confer an aggressive phenotype in papillary thyroid cancer (PTC). Clinically, the behavior of BRAF+ PTC, however, varies from an indolent to an aggressive course. SPRY2 is a negative feedback regulator of the MAPK/ERK pathway. We hypothesize that the level of SPRY2 expression contributes to MAPK/ERK pathway output and accounts for BRAF+ and clinical heterogeneity. METHODS: A tissue microarray with BRAF-positive PTCs (BRAF+ PTCs) was constructed and analyzed for SPRY2 expression and MAPK/ERK output. Data were studied in the context of clinicopathologic factors to develop a risk stratification system predictive of tumor biology. SPRY2 function was studied by silencing SPRY2 in BRAF+ PTC cells. These cells were treated with MAPK/ERK pathway inhibitors and assessed for growth effects. RESULTS: BRAF+ PTCs with an intact MAPK/ERK feedback pathway do not exhibit lymph node metastases. BRAF+ PTCs with dysregulated feedback pathways have nodal metastasis. When SPRY2 is silenced, the BRAF+ PTC cells are significantly more sensitive to MAPK/ERK inhibition. CONCLUSION: PTC behavior likely is dependent on both the driver of the MAPK/ERK pathway and its regulatory feedback. When the feedback pathway is intact, the tumor phenotype seems to be less aggressive. This observation has direct and important clinical implications and may alter our treatment strategies.

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown. Here we show that Ras-transformed cells use macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer therapies.

Given the complexity of morphological presentation and variability in clinical outcomes observed in -epithelial cancers, it is important to understand how genomic perturbations and resultant molecular aberrations lead to acquisition of tumorigenic phenotypes. Complex 3D epithelial culture systems provide investigators with the ability to propagate and manipulate primary cells in an appropriate physical setting in order to deconstruct the contribution of a given genetic lesion(s) to the process of cellular transformation. Pancreatic ductal epithelial cells (PDEC) can give rise to pancreatic intraepithelial neoplasia-precursor lesions that precede pancreatic ductal adenocarcinoma (PDA). In this chapter, we describe a series of methods for derivation and culture of primary PDEC, which can be used to elucidate the mechanistic contribution of oncogenic insults to the initiation and progression of pancreatic tumorigenesis.

Oncogenic mutations in Ras-encoding genes are found in approximately 30% of all human tumors and are most prevalent in carcinomas of the pancreas, colon, lung and bladder. Oncogenic forms of Ras are trapped in a constitutively active state and, as a consequence, promote the persistent stimulation of effector pathways that control cell growth, survival and proliferation. A prominent phenotypic feature associated with oncogenic Ras expression is the stimulation of macropinocytosis, an endocytic process that mediates the uptake of extracellular fluid via large, heterogeneous vesicles known as macropinosomes. While in amoeboid organisms this process has been linked to nutrient internalization, the functional significance of oncogenic Ras-mediated macropinocytosis is unknown. We have found that cancer cells harboring oncogenic Ras utilize macropinocytosis to internalize extracellular albumin, a major constituent of extracellular fluids and a potentially rich source of amino acids. Furthermore, we have established that tumor cells harboring oncogenic Ras mutations display robust macropinocytosis in vivo. Utilizing cell biological approaches and amino acid quantification, we determined that extracellular albumin that is internalized via oncogenic Ras-stimulated macropinosomes is targeted for degradation leading to the intracellular production of albumin-derived amino acids. Cancer cells display a heightened dependence on glutamine due to their need to sustain rapid proliferation, bioenergetics and macromolecular biosynthesis. We have observed that in cells expressing oncogenic Ras, the adverse effects of glutamine deprivation on cell growth can be rescued by macropinocytosis-mediated uptake of extracellular albumin. Moreover, the capacity of macropinocytic inhibition to diminish this growth advantage is dependent on both glutamine and glutamine-derived metabolites. These results indicate that macropinocytosis may serve as a glutamine supply mechanism that is particularly critical under conditions wher!

Ras proteins are best known to function on the plasma membrane to mediate growth factor signaling. Controlling the length of time that Ras proteins stay on the plasma membrane is an effective way to properly modulate the intensity and duration of growth factor signaling. It has been shown previously that H- and N-Ras proteins in the GTP-bound state can be ubiquitylated via a K-63 linkage, which leads to endosome internalization and results in a negative-feedback loop for efficient signal attenuation. In a more recent study, two new Ras effectors have been isolated, CHMP6 and VPS4A, which are components of the ESCRT-III complex, best known for mediating protein sorting in the endosomes. Surprisingly, these molecules are required for efficient Ras-induced transformation. They apparently do so by controlling recycling of components of the Ras pathway back to the plasma membrane, thus creating a positive-feedback loop to enhance growth factor signaling. These results suggest the fates of endosomal Ras proteins are complex and dynamic - they can be either stored and/or destroyed or recycled. Further work is needed to decipher how the fate of these endosomal Ras proteins is determined.

Mammalian cells contain three closely related ras genes, H-ras, K-ras and N-ras. Although in a given tumour type, oncogenic mutations are selectively observed in only one of the ras genes, the acquisition of the transformed phenotype has been shown to require the contribution of the normal products of the other ras genes. Here we demonstrate that oncogenic K-Ras promotes the activation of wild-type H- and N-Ras. This activation is mediated by oncogenic K-Ras-dependent allosteric stimulation of Sos and confers a growth advantage to oncogenic K-Ras harbouring cancer cells. These findings underscore the complementary functions of oncogenic and wild-type Ras in tumour cells and identify a potential new targeting strategy for Ras-driven tumours.