Rushika Perera____Lysosome Contributions to Adaptive Responses in Pancreatic Cancer Cells

Lysosomes are metabolic organelles, similar to mitochondria and peroxisomes.
PDAC is an aggressive disease affecting approximately 60,000 people each year.
The focus is on nutrient scavenging pathways like autophagy and the lysosome in PDAC.

Autophagy: A pathway that captures intracellular components (proteins, lipids, sugars, organelles) and packages them into double-membrane vesicles for lysosomal degradation.
- Alec Kimmelman's work showed autophagy is constitutively active in PDAC.
Macropinocytosis: A pathway that takes up extracellular proteins and lipids, packaging them into single-membrane vesicles for lysosomal degradation.
- Cosimo Komiso's work showed macropinocytosis is upregulated in PDAC.

Lysosomes are significantly upregulated (approximately 15-fold) in PDAC.
This upregulation is likely due to increased flux through autophagy and macropinocytosis, which converge on the lysosome.
The research aims to understand the holistic contribution of lysosomes to cancer cell adaptation and evolution within their microenvironment.

LysoIP (Lysosome Isolation by Immunoprecipitation): A technique used to isolate intact lysosomes from cells.
- Based on a method introduced by Kibant for mitochondria and established in David Sabatini's lab.
Method: Expressing tags (e.g., LAMP1, TMEM192) on the lysosome surface, conjugating them with fluorescent markers and affinity handles (e.g., TMEM192-RFP-preXHA), and then isolating lysosomes.
Allows for comparative proteomic analysis of lysosomes from different cell types (e.g., normal pancreatic ductal epithelia vs. PDAC cells).

Proteomic analysis differentiates between proteins specific to lysosomes isolated from PDAC cells and those from normal cells.
Categories of Proteins: Lysosomal resident proteins (hydrolases, transporters, enzymes, master regulators) and cargoes brought from elsewhere in the cell (e.g., via autophagy or macropinocytosis).
Analysis helps understand the lysosome's contribution to cellular renovation and metabolic homeostasis.

MHC Class I: A cell surface protein that presents antigens to the immune system.
The study identified MHC class I as being enriched in lysosomes from PDAC cells.
Hypothesis: Targeting of MHC class I to the lysosome may be a mechanism for cancer cells to evade immune detection.
In normal cells, MHC class I is on the cell membrane, while in PDAC cells, it localizes to punctate structures that overlap with autophagosomes and lysosomes.
This suggests that MHC class I is being degraded in lysosomes rather than being routed to the plasma membrane.

MHC Class I capture occurs through post-translational modification, specifically ubiquitination.
MDR1: An autophagy receptor that recognizes ubiquitinated MHC class I and brings it into a growing autophagosome, which then fuses with the lysosome for degradation.
Suppressing any step in this process (MBR1, autophagy, or lysosome) restores MHC class I on the plasma membrane and leads to a potent anti-tumor immune response in vitro and in mice.

Rush Hook System: Used to trace how MHC is trafficked in the cell.
- MHC is conjugated to GFP and a streptavidin-binding protein, attached to streptavidin linked to a hook.
- MHC is kept in the ER until exogenous biotin is added, which competes off the binding and allows MHC to traffic.
In non-PDAC cells, MHC transits through the Golgi to the plasma membrane within two hours after biotin addition.
In PDAC cells, MHC is retained in the ER even after two hours of biotin, indicating compromised trafficking.
MHC also localizes to punctate structures that colocalize with autophagosomes and lysosomes.

Hypothesis: MHC is retained in the ER and then degraded via ER-phagy (selective autophagy of the ER).
ER-phagy: A process where portions of the ER are captured by autophagy machinery and targeted for lysosomal degradation.
PDAC cells exhibit a higher rate of ER-phagy flux compared to non-PDAC cells, as measured by a CD5-pima flux reporter.
Suppression of autophagy receptors tethered to the ER (e.g., TEX264, CCPT1) affects MHC levels.
- Knockdown of TEX264 leads to an increase in MHC.
TEX264 colocalizes with MDR1, suggesting cooperation in capturing MHC class I.
Model: These receptors work together at the ER to recognize and capture ubiquitinated MHC for autophagic degradation.

Proper peptide loading onto MHC class I is essential for its trafficking and function.
MHC loading depends on the proteasome breaking down proteins into peptides in the ER and a peptide loading complex chaperoning the process.
If antigen loading is suppressed, the binding of MHC to autophagy receptors increases, enhancing clearance through autophagy.
Conversely, if cells are given a high-affinity peptide, the localization of MHC to lysosomes decreases, and more MHC is found on the cell surface.
Preventing peptide loading leads to MHC capture by autophagy, while providing a high-quality peptide allows MHC to evade autophagy-mediated immune clearance.

A CRISPR screen was used to identify E3 ligases that regulate MHC ubiquitination.
Organelle proteomics from the CCI Biohub was utilized to determine the localization of potential E3 ligases.
NFXL1: Identified as an E3 ligase that localizes to the ER and mediates ubiquitination of MHC.
- Knockout of NFXL1 decreases MHC ubiquitination and reduces the binding between MHC class I and the autophagy capture complex.
Inhibition of ubiquitination increases MHC cell surface levels.
NFXL1 and MHC protein levels are inversely correlated.
High levels of NFXL1 are linked to poor prognosis and more aggressive disease in PDAC.

Normal Cells: Efficient peptide loading leads to MHC class I trafficking to the plasma membrane.
PDAC Cells: The absence of high-affinity peptides leads to MHC stalling in the ER, increased ubiquitination by NFXL1, and capture by an autophagy complex integrating ER-tethered autophagy receptors and cytosolic receptors like MMEL1, leading to lysosomal degradation.