How do cancer cells escape tightly controlled regulatory circuits that link their growth to extracellular signaling cues? An emerging theme in cancer biology is how, in addition to genetic alterations in signaling pathways (eg. MAPK and PI3K), cancer cells can hijack normal stress response pathways to overcome reliance on external nutrients for growth. Pancreatic adenocarcinoma (PDA) is the quintessence of an aggressive malignancy that relies on constitutive activation of stress response pathways for growth and survival, with the tumors progressing rapidly in the context of extreme hypoxia, poor vascularity, and limited nutrient availability. As such, PDA employ profoundly altered networks of biosynthetic and catabolic pathways, including constitutive activation of autophagy (cellular self-catabolism) and macropinocytosis (bulk uptake of extracellular proteins), which are necessary to maintain metabolic homeostasis and drive tumorigenesis. While, these pathways are essential for tumor growth, the precise mechanism of autophagy-lysosome activation and how this organellar system contributes to metabolic reprogramming in PDA were unknown.

MiT/TFE TRANSCRIPTION FACTORS ARE OVEREXPRESSED IN PDA AND REGULATED THE EXPRESSION OF AUTOPAHGY-LYSOSOME GENES.

We now show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family transcription factors; MITF, TFE3 and TFEB. These factors show increased expression in primary human PDA tumor datasets compared to matched normal tissue and correlate with expression of a coherent network of autophagy-lysosome genes, incorporating essential autophagy genes, structural lysosomal proteins, hydrolases, solute transporters and luminal enzymes. Importantly, autophagy-lysosome components also show elevated expression in human PDA samples and cell lines, which is dependent on MiT/TFE activity.

CONSTITUTIVE ACTIVITY OF MiT/TFE PROTEINS IS MEDIATED THROUGH ENHANCED NUCLEAR IMPORT.

In non-transformed cells, MiT/TFE factors are phosphorylated by mTORC1 and retained in the cytoplasm in an inactive state under nutrient replete conditions. However, in addition to increased expression, these factors show constitutive nuclear localization and activation in PDA. Mechanistically, we show that this occurs through increased binding to nuclear import factors (IPO) that mediates their escape from mTORC1 inhibition and efficient nuclear translocation in PDA cells. Importantly, IPO expression is up-regulated in PDA cells compared to non-transformed pancreatic epithelial cells and other types of pancreatic tumors (pancreatic neuroendocrine tumor; PNET). Moreover, loss of IPO inhibits nuclear localization of MiT/TFE factors specifically in PDA cells. Therefore this work uncovers novel mechanisms enabling sustained activation of catabolic processes in PDA cells, that are only transiently induced by stress in normal cells, which incorporate alterations in the expression of tumorigenic transcription factors that act in concert with elevated levels of nuclear import proteins.

MiT/TFE FACTORS FUNCTION TO MAINTAIN LYSOSOME INTEGRITY IN PDA CELLS.

Having clarified the mechanisms of their constitutive nuclear import, we turned to the functional roles of MiT/TFE factors in PDA. Our transcriptional data imply contributions of the activated MiT/TFE proteins to the integrity and function of the lysosomal system in PDA cells. To test this key point, we depleted MiT/TFE proteins in PDA cells and observed striking defects in lysosome morphology, degradation of cargo protein and maturation of autophagosomes. In addition, we found that loss of MiT/TFE factors in PDA cells led to a dramatic defect in lysosomal pH. Importantly, these parameters were not altered following knockdown of MiT/TFE proteins in non-transformed control cell lines. Reciprocally, MITF or TFE3 overexpression in HPDE, HPNE, or PDA cells induced autophagy-lysosomal gene expression, LC3B foci, and lipidated LC3-II that was further enhanced following treatment with chloroquine (CQ), an inhibitor of lysosome acidification, indicating a marked augmentation of autophagic flux. Collectively, these data show that MiT/TFE proteins govern both autophagic flux and lysosome activity in PDA cells. This integrated cellular clearance program appears to enable efficient processing of cargo from autophagy as well as macropinocytosis, providing PDA cells access to critical sources of both intracellular and extracellular nutrients.

AMINO ACIDS ARE THE PRIMARY METABOLITE POOL DERIVED FROM ENHANCED AUTOPHAGY-LYSSOOME ACTIVITY IN PDA.

By degrading numerous cellular substrates, the lysosome generates metabolic intermediates that may feed into multiple pathways. We took advantage of the impaired autophagy-lysosome function caused by MiT/TFE inactivation to dissect the metabolic circuitry that sustains PDA growth. First, we conducted global metabolite profiling of PDA cells transfected with control or TFE3-targeted siRNAs. Dual gas chromatography and mass spectrometry (GC/MS) and liquid chromatography mass spectrometry (LC/MS) detected 347 known metabolites. Of these, 15.2% (53/347) showed a statistically significant change upon TFE3 inactivation in both cell lines (48/53 downregulated, 5/53 upregulated). Most prominently altered were amino acids (AA) and their breakdown products, with 31% (25/80) showing decreased abundance. No change in AA uptake was observed upon TFE3 silencing suggesting that in PDA the autophagy-lysosome system may supply a significant fraction of intracellular AA irrespective of external availability. Correspondingly, TFE3 inactivation or Bafilomycin A1 treatment (a specific inhibitor of the lysosomal V-H+ATPase), caused a significant decrease in intracellular AA levels in a multiple PDA cell lines, as did specific inactivation of autophagy by ATG5 knockdown. Importantly, these manipulations did not result in significant changes in AA levels in control non-PDA cells. Thus, the MiT/TFE factors and the autophagy-lysosome system are critical and specific regulators of intracellular AA abundance in PDA.

MiT/TFE FACTORS ARE REQUIRED FOR PDA GROWTH IN VITRO AND IN VIVO.

Consistent with their key roles in organelle function and metabolic regulation, the MiT/TFE proteins were central to PDA growth. PDA cell lines expressing high endogenous levels of MITF, TFE3, or TFEB were exceedingly sensitive to knockdown of that factor, displaying marked impairment in colony formation and reduction in proliferation, whereas non-PDA control cells were unaffected. Expression of shRNA-resistant cDNAs of MITF and TFE3 rescued proliferation in the knockdown setting, confirming the specificity of these experiments. Furthermore, PDA cells were broadly sensitive to chloroquine treatment (CQ; a well characterized autophagy inhibitor) as compared to non-PDA control cells.

Conversely, ectopic expression of MITF or TFE3 rendered control cells hypersensitive to CQ treatment, linking MiT/TFE-regulated clearance pathways to these growth phenotypes. We next sought to test the contributions of MiT/TFE to PDA tumorigenicity in vivo. Significantly, TFE3 or MITF knockdown virtually abolished xenograft tumor growth of PDA cells. In reciprocal gain-of-function studies, we investigated the impact of MITF overexpression on the tumorigenicity of primary KrasG12D-expressing mouse ductal epithelial cells. KrasG12D control cells formed only focal low-grade PanIN-like lesions by six weeks following orthotopic injection, whereas co-expression of MITF induced the expression of autophagy-lysosome genes and resulted in large orthotopic tumors in mice. In summary, our work places a new focus on lysosome regulation by MiT/TFE proteins as a nexus for metabolic reprogramming in PDA cells. Beyond serving a generic housekeeping role, lysosomes show increased activity in PDA and are critical integrators of major routes for nutrient scavenging and metabolic adaptation, providing an alternate pathway for maintaining intracellular AA stores. These studies also demonstrate activation of clearance pathways converging on the lysosome as a novel hallmark of aggressive malignancy.

Citation Format: Rushika M. Perera, Svetlana Stoykova, Brandon N. Nicolay, Kenneth N. Ross, Julien Fitamant, Myriam Boukhali, Vikram Deshpande, Martin K. Selig, Cristina R. Ferrone, Jeff Settleman, Gregory Stephanopoulos, Nicholas J. Dyson, Roberto Zoncu, Sridhar Ramaswamy, Wilhelm Haas, Nabeel M. Bardeesy. Transcriptional mechanisms for autophagy regulation and metabolic reprogramming in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG08. doi:10.1158/1538-7445.AM2015-NG08

©2015 American Association for Cancer Research.
2015