Inhibiting cholesterol uptake decreases growth of pancreatic tumors [PreClinical]

1. Metabolic profiling of cells from mice bearing spontaneous pancreatic ductal adenocarcinoma (PDAC) revealed high upregulation of genes relating to cholesterol synthesis and storage as compared to control cells.

2. Specific silencing of the low density lipoprotein receptor (LDLR), which mediates cellular cholesterol increases, resulted in decreased tumor cell proliferation in vitro, and in vivo when combined with traditional chemotherapy treatment.

Evidence Rating Level: 3 (Average)

Study Rundown: PDAC remains a particularly deadly form of cancer, with a median survival rate of less than 6 months. Effective treatment of PDAC remains elusive, and new approaches are critically needed. As it is well known that tumors undergo metabolic reprogramming, the authors of this study aimed to develop a PDAC treatment that catered toward the PDAC metabolic signature. Metabolic gene expression was compared between tumor tissue isolated from PDAC-bearing mice and pancreatic tissue from control mice. Striking differences were identified between the two datasets, including the upregulation of lipoprotein- and cholesterol-related genes in PDAC tissue as compared to controls. Subsequent protein expression analysis showed PDAC tissue had high levels of LDLR, which plays a role in cholesterol uptake in cells. While previous studies have failed to slow PDAC growth by blocking cholesterol synthesis, this is the first investigation of blocking cholesterol uptake to inhibit PDAC survival.

To investigate the role of LDLR in cancer survival and growth researchers silenced the receptor in PDAC cells (shLdlr). These shLdlr cells exhibited decreased proliferation as compared to controls and were more sensitive to gemcitabine (GEM), the current chemotherapeutic of choice for treating PDAC. Mice injected with shLdlr cells and treated with GEM showed significantly lowered tumor growth and endpoint tumor weight than identically treated mice injected with control PDAC cells. Using clinical tissue samples, researchers found human PDAC samples showed significant correlation between low LDLR gene expression and disease-free survival.

Translating the gene-silencing approach outlined in this study to human PDAC therapy will not be straightforward. Development of gene therapies can be particularly complicated, and the authors do not explore a pharmacological mechanism for targeting LDLR. That noted, this work identifies LDLR as a novel target for PDAC treatment, which may eventually result in improved patient outcomes.

Click to read the study in PNAS

Relevant Reading: Elevated uptake of low density lipoprotein by drug resistant human leukemic cell lines

In-Depth [in vitro and animal study]: Pancreatic tumor and tissue samples from mice bearing spontaneous PDAC tumors and from control littermates were extracted for analysis after sacrifice. DNA microarray technology identified differences in metabolic RNA transcript expression between control and PDAC tissue. Of the 2,177 metabolic genes analyzed, 427 were upregulated and 320 were downregulated in PDAC as compared to control. Significantly upregulated genes included genes relating to cholesterol synthesis, storage and uptake (n=3, p<0.05). The LDLR gene exhibited 8.2-fold increased expression in PDAC as compared to controls, and western blotting showed corresponding LDLR protein expression to be enhanced 7.7-fold (p<0.05).

LDLR gene silencing was accomplished using small hairpin RNAs (shRNA). Cells established from the PDAC mice and expressing shRNA targeting a particular LDLR gene sequence (shLdlr3) showed a 70% reduction in LDLR protein as compared to control (p<0.05). Proliferation of shLdlr3 cells was also lower than of control cells (p<0.05). When subjected to GEM treatment, ShLdlr3 cells exhibited lowered viability, and had a half-inhibitory concentration of 7.4±1.2 nM as compared to 13.8±0.4 nM in control cells. In vivo experiments involved implanting mice with shLdlr3 or control PDAC cells and administering GEM twice weekly beginning a week after tumor establishment. Tumor size at time of analysis was significantly smaller in shLdlr3 than in control mice (p<0.05, n=10 per group).

Finally, tumor specimens from human patients were examined. LDLR gene expression did not correlate with tumor stage. However, survival curves showed that low LDLR expression correlated with disease-free survival (p=0.008, n=10 for high LDLR, n=13 for low LDLR), but not overall survival (p =0.201).

Image: PD

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