Macropinocytosis Fuels Prostate Cancer

Cancer cells utilize various nutrient acquisition pathways to fulfill their metabolic demands, which notably include macropinocytosis, an amino acid supply pathway that supports tumor cell survival and proliferation (1). This evolutionarily conserved fluid phase form of bulk endocytosis internalizes and targets extracellular proteins for lysosomal degradation, thereby generating protein-derived amino acids to augment cellular metabolism. This form of nutrient scavenging appears to be particularly critical for sustaining intracellular amino acid pools that are in short supply due to the nutrient-poor conditions of the tumor microenvironment (1). Initial studies on the role of macropinocytosis in tumor biology focused on serum albumin uptake as a nutrient source, as it represents the most abundant protein in physiologic fluids and normally accounts for approximately 50% of the protein content in human plasma (2). Although albumin degradation can indeed serve as a rich source of amino acids, it is important to recognize that albumin physiologically functions as a carrier protein. Hence, various additional albumin-bound molecules can presumably enter tumor cells via macropinocytosis, including most notably fatty acids and cholesterol, both of which are important to the production and integrity of cellular membranes, respectively. In this way, the macropinocytosis of serum albumin has the capacity to support the metabolic and biosynthetic demands of the tumor cell on many levels. In this issue of Cancer Discovery, Kim and colleagues delineate how necrotic cell debris, a previously unappreciated macropinocytic cargo, similarly supports prostate cancer cell metabolism and tumor growth in PTEN-deficient prostate cancer cells (3).

Kim and colleagues establish that PTEN loss drives constitutive macropinocytosis in prostate cancer cells, and that PTEN reconstitution can suppress this endocytic pathway. This is consistent with the established role of PTEN as a lipid phosphatase that counteracts PI3K signaling, which is integral to macropinocytic induction (Fig. 1). Intriguingly, although macropinocytosis in these PTEN-deficient prostate cancer cells is not enhanced by nutrient deprivation, expression of a dominant-negative form of AMPK, a kinase that acts as a cellular energy sensor, partially abrogates macropinosome dynamics and suppresses proliferation in nutrient-limiting conditions. Remarkably, AMPK activation also induces autophagy, another nutrient-scavenging pathway complementary to macropinocytosis, by delivering intracellular macromolecules to the lysosome (4). However, upon genetically interrogating these two lysosomal pathways in PTEN-deficient cells, Kim and colleagues delineate a critical requirement for macropinocytosis, but not autophagy, in promoting AMPK-driven proliferation in low-nutrient conditions (3). Indeed, an important avenue for further study is determining to what extent these effects of AMPK on macropinocytosis affect cell fitness in other oncogenic contexts and cancer types.

In oncogenic RAS-expressing cells, albumin is exclusively internalized by macropinocytosis (2). In contrast, this study reveals that albumin uptake can still occur in PTEN-deficient prostate cancer cells when macropinocytosis is inhibited using 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a pharmacologic macropinocytosis inhibitor. In further support of this hypothesis, whereas protein-derived amino acids generated by the macropinocytosis pathway can activate mTOR signaling in cells transformed with oncogenic RAS (5), inhibiting macropinocytosis with EIPA in prostate cancer cells only partially attenuates mTOR signaling. These data suggest that a portion of extracellular albumin is internalized by macropinocytosis in PTEN-deficient prostate cancer cells, but that other endocytic pathways also contribute to its internalization. There are at least seven membrane-associated albumin-binding proteins, many of which, including FcRn, cubilin, megalin, and calreticulin, are internalized by receptor-mediated endocytosis. It is plausible that one or more of these albumin receptors mediates albumin uptake in prostate tumor cells; further research is required to assess whether and how such pathways function more broadly in tumor cell nutrient acquisition.

Unlike albumin uptake, which does not absolutely depend on macropinocytosis in prostate cancer cells, Kim and colleagues identify necrotic debris as an important macropinocytic cargo that is catabolized to generate protein-derived amino acids and build biomass. Moreover, engulfment of membranes and lipids present in necrotic debris helps maintain lipid stores in prostate cancer cells. With such a nutrient-rich cargo, it will be interesting to further scrutinize whether lysosomal breakdown of necrotic debris provides a broader array of building blocks, not just amino acids and lipids, to tumor cells.

The macropinocytic internalization of smaller necrotic debris, and not larger live or apoptotic cells, is consistent with the characteristics of this uptake pathway. The large discrete vesicles formed during macropinocytosis, known as macropinosomes, range in size from 0.2 up to 2–5 μm and are nonselective in terms of cargo. In fact, macropinocytic uptake likely facilitates the entry of a plethora of nourishing substances in addition to albumin, including abundant serum proteins such as globulins and fibrinogen. The hydrolysis of these proteins may also serve as an amino acid source to support cancer cell metabolism. In addition to serum proteins, extracellular matrix (ECM) proteins are also internalized into tumor cells by macropinocytic uptake (6, 7). Collagen fragments are taken up by pancreatic ductal adenocarcinoma (PDAC) cells, and the breakdown of these fragments provides collagen-derived proline to promote PDAC cell survival under nutrient-limiting conditions. Fibronectin, a high-molecular weight glycoprotein found in the ECM, also enters PDAC cells via macropinocytosis and, once catabolized, may represent a source of not only amino acids, but also carbohydrates. Finally, macropinocytosis serves as an internalization mechanism for extracellular organelles, such as exosomes and microvesicles, involved in intercellular communication (8). Uptake of these extracellular vesicles into tumor cells may facilitate the delivery of their contents to the cytoplasm or represent a means of clearing these vesicles from the extracellular space. Irrespective of the functional consequences of internalizing these organelles by macropinocytosis, the lysosomal breakdown of such vesicular cargo can provide nutrients to rapidly proliferating tumor cells. Finally, because macropinosomes encapsulate extracellular fluid, and anything solubilized therein, electrolytes and small molecules such as amino acids and glucose are likely to gain access to the cell through macropinocytosis (9). In fact, even the water molecules internalized in this fashion are thought to play an important role in cell volume control.

An important technological breakthrough in Kim and colleagues’ work is the development of novel isotopic labeling strategies to scrutinize protein scavenging in cancer cells (3). Previously, one of two methods has been employed to evaluate the extent to which proteins internalized by macropinocytosis contribute to intracellular amino acid pools and biomass. The first method involves feeding tumor cells a soluble, heat-inactivated, ¹³C-labeled yeast protein extract, which is subsequently quantified in the extracted intracellular metabolites using mass spectrometry (2). This method has proven useful to ascertain the contribution of protein-derived amino acids to intracellular amino acid pools as well as the ability of protein-derived amino acids to contribute to central carbon metabolism, as evidenced by ¹³C-labeling of pyruvate, lactate, and various tricarboxylic acid cycle metabolites. Partially labeled mass isotopomers in numerous metabolites have suggested that protein-derived amino acids are being metabolized through several pathways, including glutamine anaplerosis/oxidation, acetyl-coenzyme A metabolism, reductive carboxylation, and serine/glycine cycling. The second method essentially reverses the isotope labeling read-out such that protein-derived amino acids are unlabeled and quantified in relation to an intracellular ¹³C- and ¹⁵N-labeled metabolite pool (10). Near-complete intracellular metabolite labeling is established by growing tumor cells in media containing only uniformly ¹³C- and ¹⁵N-labeled glucose and amino acids. Tumor cells are then fed unlabeled BSA and metabolites extracted and analyzed by mass spectrometry. Employing this methodology corroborated that protein-derived amino acids contribute substantially to the intracellular amino acid pool and that most amino acid nitrogen is predominantly derived from extracellular protein. The new method described in Kim and colleagues’ work employs necrotic cellular debris labeled using “stable isotope labeling with amino acids in cell culture” (SILAC) as a food source for unlabeled prostate cancer cells, upon which the extent of label incorporation into intracellular peptides is assessed via mass spectrometry. FL5.12 hematopoietic cells are used as a source of labeled necrotic debris by growing them in SILAC medium containing ¹³C₃, ¹⁵N₁ lysine and ¹³C₆, ¹⁵N₄ arginine for an extended number of generations prior to the induction of necrotic cell death. In protein extracts prepared from prostate cancer cells grown in unlabeled medium but fed labeled necrotic debris, the resulting tryptic peptides containing both lysine and arginine can be present in different isotopic forms. These peptides include those unlabeled and generated independently of macropinocytosis, those that contain both unlabeled and labeled amino acids, and fully isotopically labeled peptides. Peptides containing both unlabeled and labeled amino acids most likely represent those generated using macropinocytosis-derived amino acids, as fully labeled peptides could have originated from undigested, heavy-labeled FL5.12 proteins. By evaluating the extent of peptide labeling, this approach allowed for the determination that up to 70% of the biomass and the intracellular amino acid pool in these prostate cancer cells can be derived from macropinocytosis. Because increased tumor necrosis in humans is commonly associated with increased aggressiveness and poor prognosis, an intriguing question for future study is to analyze how macropinocytosis of necrotic debris promotes tumor biomass in both prostate and other solid cancers in vivo. On the whole, Kim and colleagues greatly expand our understanding of this nutrient-scavenging pathway downstream of PI3K activation and further reinforce macropinocytosis as a central fuel source required for cancer cell growth.

J. Debnath is a consultant at 5AM Ventures, LLC, and is a consultant/advisory board member for Vescor Therapeutics, LLC. No potential conflicts of interest were disclosed by the other author.