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IN THE SPOTLIGHT

Macropinocytosis fuels Prostate Cancer Cosimo Commisso 1 and Jayanta Debnath 2

1 Tumor Initiation and Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. 2 Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. Corresponding Authors: Jayanta Debnath, University of California, San Francisco, 513 Parnassus Avenue, HSW450B, San Francisco, CA 94143. Phone: 415-476-1780; Fax: 414-514-3165; E-mail: jayanta.debnath@ucsf.edu ; and Cosimo Commisso, Sanford Burnham Prebys Medical Dis-covery Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037. Phone: 858-795-5171; E-mail: ccommisso@sbpdiscovery.org doi: 10.1158/2159-8290.CD-18-0513 ©2018 American Association for Cancer Research.

summary: Kim and colleagues identify necrotic debris as a macropinocytic cargo in PTEN-defi cient prostate cancer cells, which is catabolized to generate the nutrients and biomass necessary to support tumor cell growth and metabolism in nutrient-limiting conditions. Cancer Discov; 8(7); 800–2. ©2018 AACR.

See related article by Kim et al., p. 866 (3) .

Cancer cells utilize various nutrient acquisition pathways to fulfi ll their metabolic demands, which notably include macropinocytosis, an amino acid supply pathway that sup-ports tumor cell survival and proliferation ( 1 ). This evolu-tionarily conserved fl uid 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 scav-enging appears to be particularly critical for sustaining intra-cellular 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 physi-ologic fl uids 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 pre-viously unappreciated macropinocytic cargo, similarly sup-ports prostate cancer cell metabolism and tumor growth in PTEN-defi cient prostate cancer cells ( 3 ).

Kim and colleagues establish that PTEN loss drives con-stitutive 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 ). Intrigu-ingly, although macropinocytosis in these PTEN-defi cient prostate cancer cells is not enhanced by nutrient depriva-tion, expression of a dominant-negative form of AMPK, a kinase that acts as a cellular energy sensor, partially abro-gates macropinosome dynamics and suppresses proliferation in nutrient-limiting conditions. Remarkably, AMPK activa-tion 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-defi cient cells, Kim and colleagues delineate a criti-cal requirement for macropinocytosis, but not autophagy, in promoting AMPK-driven proliferation in low-nutrient condi-tions ( 3 ). Indeed, an important avenue for further study is determining to what extent these effects of AMPK on macro-pinocytosis affect cell fi tness 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-defi cient prostate cancer cells when macropinocytosis is inhibited using 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a pharmaco-logic macropinocytosis inhibitor. In further support of this hypothesis, whereas protein-derived amino acids generated by the macropinocytosis pathway can activate mTOR signal-ing 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-defi cient 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 recep-tor-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 macropi-nocytic cargo that is catabolized to generate protein-derived

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amino acids and build biomass. Moreover, engulfment of membranes and lipids present in necrotic debris helps main-tain 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, macropi-nocytic 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 adeno-carcinoma (PDAC) cells, and the breakdown of these frag-ments 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 cat-abolized, 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

Figure 1.  Macropinocytosis in PTEN-deficient prostate cancer cells drives the uptake of necrotic cell debris to support tumor cell fitness. PTEN loss enhances receptor tyrosine kinase (RTK)–mediated PI3K signaling, which, together with AMPK-dependent RAC1 activity, stimulates the macropinocytic uptake of necrotic cell debris. Lysosome-dependent degradation of this necrotic debris produces protein-derived amino acids and other nutrients that contribute to the intracellular nutrient pool, biomass production, and mTOR activation. Image has been modified from refs. 1, 3.

Necrotic celldebris

PI3K RAC1

Lysosome

Amino acidsLipids

Other nutrients

mTOR

Biomass

PI(4,5)P2

PTEN

AMPK

PI(3,4,5)P3

RTK

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consequences of internalizing these organelles by macropino-cytosis, 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 col-leagues’ 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 macro-pinocytosis 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 pyru-vate, 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, reduc-tive 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 con-taining 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 cul-ture” (SILAC) as a food source for unlabeled prostate cancer cells, upon which the extent of label incorporation into intra-cellular 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 result-ing tryptic peptides containing both lysine and arginine can be present in different isotopic forms. These peptides include those unlabeled and generated independently of macropino-

cytosis, those that contain both unlabeled and labeled amino acids, and fully isotopically labeled peptides. Peptides contain-ing both unlabeled and labeled amino acids most likely repre-sent 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 deter-mination 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 down-stream of PI3K activation and further reinforce macropino-cytosis as a central fuel source required for cancer cell growth.

Disclosure of Potential Conflicts of InterestJ. Debnath is a consultant at 5AM Ventures, LLC, and is a consult-

ant/advisory board member for Vescor Therapeutics, LLC. No poten-tial conflicts of interest were disclosed by the other author.

Published online July 2, 2018.

RefeRenCes 1. Recouvreux MV, Commisso C. Macropinocytosis: a metabolic adapta-

tion to nutrient stress in cancer. Front Endocrinol 2017;8:261. 2. Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kam-

phorst JJ, Hackett S, et al. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 2013;497:633–7.

3. Kim SM, Nguyen TT, Ravi A, Kubiniok P, Finicle BT, Jayashankar V, et  al. PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells. Cancer Discov 2018;8:866–83.

4. Kimmelman AC, White E. Autophagy and tumor metabolism. Cell Metab 2017;25:1037–43.

5. Palm W, Park Y, Wright K, Pavlova NN, Tuveson DA, Thompson CB. The utilization of extracellular proteins as nutrients is suppressed by mTORC1. Cell 2015;162:259–70.

6. Davidson SM, Jonas O, Keibler MA, Hou HW, Luengo A, Mayers JR, et al. Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors. Nat Med 2017;23:235–41.

7. Olivares O, Mayers JR, Gouirand V, Torrence ME, Gicquel T, Borge L, et al. Collagen-derived proline promotes pancreatic ductal adenocar-cinoma cell survival under nutrient limited conditions. Nat Commun 2017;8:16031.

8. Nakase I, Kobayashi NB, Takatani-Nakase T, Yoshida T. Active macropinocytosis induction by stimulation of epidermal growth fac-tor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes. Sci Rep 2015;5:10300.

9. Yoshida S, Pacitto R, Yao Y, Inoki K, Swanson JA. Growth factor sign-aling to mTORC1 by amino acid-laden macropinosomes. J Cell Biol 2015;211:159–72.

10. Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, et  al. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein. Cancer Res 2015;75:544–53.

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