LKB1 is a central regulator of tumor initiation and pro-growth metabolism in ErbB2-mediated breast cancer

Abstract

Background: Germline and somatic mutations in STK11, the gene encoding the serine/threonine kinase LKB1, are strongly associated with tumorigenesis. While loss of LKB1 expression has been linked to breast cancer, the mechanistic role of LKB1 in regulating breast cancer development, metastasis, and tumor metabolism has remained unclear.

Methods: We have generated and analyzed transgenic mice expressing ErbB2 in the mammary epithelium of LKB1 wild-type or LKB1-deficient mice. We have also utilized ErbB2-expressing breast cancer cells in which LKB1 levels have been reduced using shRNA approaches. These transgenic and xenograft models were characterized for the effects of LKB1 loss on tumor initiation, growth, metastasis and tumor cell metabolism.

Results: We demonstrate that loss of LKB1 promotes tumor initiation and induces a characteristic shift to aerobic glycolysis ('Warburg effect') in a model of ErbB2-mediated breast cancer. LKB1-deficient breast cancer cells display enhanced early tumor growth coupled with increased cell migratory and invasive properties in vitro. We show that ErbB2-positive tumors deficient for LKB1 display a pro-growth molecular and phenotypic signature characterized by elevated Akt/mTOR signaling, increased glycolytic metabolism, as well as increased bioenergetic markers both in vitro and in vivo. We also demonstrate that mTOR contributes to the metabolic reprogramming of LKB1-deficient breast cancer, and is required to drive glycolytic metabolism in these tumors; however, LKB1-deficient breast cancer cells display reduced metabolic flexibility and increased apoptosis in response to metabolic perturbations.

Conclusions: Together, our data suggest that LKB1 functions as a tumor suppressor in breast cancer. Loss of LKB1 collaborates with activated ErbB2 signaling to drive breast tumorigenesis and pro-growth metabolism in the resulting tumors.

Figure 1

LKB1 loss promotes the initiation of ErbB2-induced mammary tumors. (A) Kaplan-Meier analysis, depicting the percentage of tumor-free animals over time in NIC/LKB1^+/+and NIC/LKB1^fl/fl cohorts. The T₅₀ values represent the time at which 50% of the mice develop their first palpable mammary tumor. n, number of animals analyzed in each cohort. (B) Number of tumor-bearing mammary glands in each cohort. The average number of involved glands is increased in NIC/LKB1^fl/fl (7.5 ±1.1) compared with NIC/LKB1^+/+ mice (5.4 ±1.4) (***, P< 0.001). (C) Hematoxylin staining of mammary gland whole mounts dissected from 3-month-old NIC/LKB1^+/+ and NIC/LKB1^fl/fl mice. Arrows indicate the presence of pre-neoplastic lesions. (D) Mammary tumor growth following mammary fat pad injection of NIC tumor cells harboring shRNAs targeting FireFly luciferase (NIC-FF) and NIC mammary tumors with stable LKB1 knockdown (NIC-LKB1 KD). NIC-FF or NIC-LKB1 KD cells were injected in the mammary fat pads of 8-week old SCID/beige mice (n = 10 mice per group) and tumor growth was monitored by bi-weekly calliper measurements (*, P< 0.05).

Figure 2

LKB1 knockdown in breast cancer cells causes reduced expression of epithelial markers and acquisition of migratory and invasive properties. (A) Representative immunofluorescent images of NIC-FF and NIC-LKB1 KD cells in 3D collagen cultures stained with E-cadherin (red) and ZO-1 (purple). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue) and GFP images are shown to confirm that breast cancer cells retain expression of the control or LKB1-targeting shRNAs. The scale bar in the upper left inset represents 20 μm and applies to all panels. (B) Quantification of the number of cell colonies exhibiting strong junctional protein expression (E-cadherin and ZO-1 staining). The data correspond to an average of three independent experiments. **, P< 0.01. The migratory (C) and invasive (D) rates of NIC-FF and NIC-LKB1 KD cells were assessed using the xCELLigence platform. The data represent an average of three independent experiments performed in duplicate. *, P< 0.05.

Figure 3

Loss of LKB1 results in reduced lung metastatic burden. The percentage of mice within each cohort that developed spontaneous lung metastases, the number of lung metastases, the average lesion size and the percentage lesion area present per total lung area were determined at necropsy on mice subjected to mammary fat pad injection with NIC-FF and NIC LKB1 KD cells (n = 10 mice per group) (A-D) or mice subjected to tail vein injections with NIC-FF and NIC LKB1 KD cells (n = 10 mice per group) (E-H). *, P< 0.05; **, P< 0.01; ***, P< 0.001. n.s., not significant.

Figure 4

LKB1 loss confers a pro-growth signal transduction signature in ErbB2-positive mammary tumors. (A) Five NIC/LKB1^+/+and five NIC/LKB1^fl/fl mammary tumors were subjected to RPPAanalysis. Expression of selected proteins and phospho-proteins that are differentially expressed between NIC/LKB1^+/+ and NIC/LKB1^fl/fl mammary tumors. Color key indicates level of expression, with green signifying proteins and phospho-proteins that are underexpressed and red identifying those that are overexpressed compared with control cells. (B) Immunoblot analysis of mammary tumor lysates derived from NIC/LKB1^+/+and NIC/LKB1^fl/fl mice with antibodies directed to components of the mTOR and Akt signaling pathways. Immunoblot analysis for β-actin serves as a loading control.

Figure 5

Loss of LKB1 results in increased bioenergetic markers in ErbB2-positive mammary tumors and derived cell lines. (A-F) Six NIC/LKB^+/+ and six NIC/LKB1^fl/fl mammary tumors were subjected to LC-MS analysis. Intracellular levels of glucose (A), lactate (B), creatine (C), ATP (D), ADP (E), and AMP (F) are represented as μM per mg of tumor. (G-J) NIC-FF and LKB1 KD cells were subjected to metabolic analyses as in (A-F). Intracellular levels of ATP (G), ADP (H) and AMP (I) are represented in μM per 10⁶ cells. The AMP:ATP ratio (J) in LKB1 KD cells is expressed relative to the ratio for control cells. *, P< 0.05; **, P< 0.01.

Figure 6

LKB1-deficient breast cancer cells display increased aerobic glycolysis. (A-B) Extracellular flux analysis of NIC cell lines. NIC-FF and NIC-LKB1 KD cells were plated for ECAR (A) and OCR (B) analyses. The data represent one representative experiment of three independent replicates and the values correspond to an average of five wells per experiment. (C-D) NIC-FF and NIC-LKB1 KD cells were cultured for 48 hours and the relative extracellular (C) and intracellular (D) levels of lactate were determined using an enzymatic assay and GC-MS, respectively. The data represent one representative experiment from three independent replicates, each performed in triplicate. (E) Total RNA was isolated from NIC-FF and NIC-LKB1 KD cells, and the relative mRNA expression of several glycolytic enzymes was determined by qPCR. Transcript levels were determined relative to Rpl13 (60S ribosomal protein L13) mRNA levels, and normalized relative to its expression in control NIC-FF cells. The data represent the average of three independent experiments, each performed in triplicate. (F) Glucose uptake in NIC-FF and NIC-LKB1 KD cells was measured by flow cytometry using the 2NBDG fluorescent glucose analog. The data correspond to one representative experiment out of four independent replicates, each performed in triplicate. *, P< 0.05; **, P< 0.01; ***, P< 0.001.

Figure 7

Loss of LKB1 sensitizes cells to metabolic stress. (A) Immunoblotting was performed on protein extracts from NIC-FF and NIC-LKB1 KD cells treated with rapamycin (100 nM) for 24 hours to confirm mTOR activity. Arrows point to the hypophosphorylated forms of 4E-BP1. (B-C) ECAR analysis of NIC-FF and NIC-LKB1 KD cells treated or not with rapamycin for 24 hours in full glucose conditions (B) or in 1 mM glucose conditions (C). (D) ECAR analysis of NIC-FF and NIC-LKB1 cells treated with vehicle or with metformin (5 mM) for 6 hours. (E-F). Viability assays were performed on NIC-FF and NIC-LKB1 KD cells cultured in 25 mM glucose or 1 mM glucose and treated or not treated with rapamycin for 72 hours. The level of apoptosis was assessed using 7-AAD (E) or cleaved caspase 3 levels (F). All graphs correspond to a representative experiment of three performed, and values represent the average of six wells for (B), (C), (D) and (F) and three wells for (E). (G) NIC-FF and NIC-LKB1 KD cells were cultured for 24 hours in 1 mM glucose and intracellular ATP levels were quantified as the percentage drop from baseline conditions (25 mM of glucose). *, P< 0.05; **, P< 0.01; ***, P< 0.001.