LKB1 is a central growth-regulatory kinase that exerts its effects, in part, through the negative regulation of pro-growth pathways such as mTOR. LKB1 is a well-established tumor suppressor, with both germline and somatic mutations in STK11, the gene encoding LKB1, associated with cancer development. While broadly linked with cancer, LKB1’s role in breast cancer development and metabolic regulation in primary tumors has been poorly understood. To address this question, we created a genetically engineered mouse model to assess the impact of LKB1 deletion on the development and progression of breast tumors driven by the ErbB2 oncogene. We observed that ErbB2-mediated breast tumorigenesis is enhanced by LKB1 deletion, which is consistent with both experimental and clinical data linking LKB1 to breast cancer [41, 42]. Recent work by Andrade-Vieira and colleagues  also investigated the role of LKB1 in ErbB2-mediated tumorigenesis using a similar mouse strain (stk11
−/−; NIC). They observed a ~ 25% decrease in tumor latency, which was not apparent in our model. We observed a ~ 8% reduction in median tumor latency that was not statistically significant (Figure 1A). However, detailed whole-mount analysis of our mice revealed the presence and early onset of hyperplastic lesions in the mammary epithelium when LKB1 was absent (Figure 1C). This observation was similar to the growth advantage displayed by LKB1-knockdown NIC tumor cells in vivo at early time points (Figure 1D). Thus, despite differences in tumor latency, both models indicate that LKB1 loss can cooperate with ErbB2 to promote breast tumor initiation. Our data suggest that the prominent phenotypic changes associated with LKB1-deletion in breast tumors are a reprogramming of signal transduction and metabolic pathways to favor increased bioenergetic capacity and cell growth.
Our work and that of other groups  suggests that loss of LKB1 cooperates with oncogenes to modulate the initiation and growth properties of tumors; however, our data indicate that the impact of reducing LKB1 expression on breast cancer development is complex. LKB1 deletion in mammary tissue promotes the induction of mammary tumors with low penetrance and long latency , suggesting that LKB1 deletion or loss-of-heterozygosity may not be a significant driving event for breast cancer. In contrast, deletion of STK11 in MYC-driven breast tumor models significantly reduced the latency period for tumor development . In the context of tumors driven by unregulated ErbB2 signaling, complete loss of LKB1 does not affect the latency of tumor formation driven by the ErbB2 oncogene; rather, it increases the total number of pre-neoplastic lesions and overt tumors that form in these animals. Thus, while ErbB2 may drive the establishment of primary tumors, increased cell growth, and deregulated metabolism, the differences observed between the ErbB2 model and that of other groups may reflect differences in the mechanisms by which ErbB2 promotes tumor development relative to other oncogenes. Both PI3K, which is activated by ErbB2, and MYC are strong drivers of metabolism; thus loss of LKB1 may synergize specifically with these oncogenes by enhancing pro-growth metabolic pathways.
One of the striking features observed in primary LKB1-deficient ErbB2-postive breast tumors is the amplification of signal transduction pathways impacting cell growth and metabolism. ErbB2 is an oncogenic receptor tyrosine kinase that initiates signaling pathways that control both cell proliferation and survival, including MAPK/ERK and PI3K/Akt pathways . Using RPPA analysis to detect changes in signal transduction pathways in primary breast tumors, we observed major shifts in cellular signaling specifically in primary LKB1-deficient ErbB2-positive breast tumors. Consistent with previously established roles for LKB1, deletion of LKB1 led to decreased AMPK signaling and increased mTORC1 signaling in ErbB2-positive breast tumors. However, we also observed evidence of enhanced signaling by other kinases including Src, MEK1, and MAPK, suggesting a previously unappreciated negative regulatory role of LKB1 on these pathways. Importantly, reducing AMPK activity may not be the only means by which mTOR activity is elevated in LKB1-deficient tumors. Akt is a direct activator of mTOR, and both Akt phosphorylation (T308/S473) and phosphorylation of Akt targets (GSK3β, PRAS40) were elevated in ErbB2-positive tumors lacking LKB1. By removing an endogenous repressor of both mTOR and Akt activity, LKB1 loss may be one way for oncogenic ErbB2 to reprogram signal transduction in tumors to promote metabolism and increased cell growth during transformation.
One of the mechanisms by which oncogenes promote tumor cell growth and proliferation is through enhanced activation of key metabolic pathways, such as glycolysis . Here we show that loss of LKB1 in ErbB2-mediated breast cancer is sufficient to promote the Warburg effect. ErbB2-positive breast cancer cells lacking LKB1 displayed increased expression of several enzymes and transporters that support glycolysis, and both glycolytic flux and overall lactate production were enhanced in LKB1-deficient breast cancer cells. The enhanced glycolytic metabolism observed in LKB1-deficient breast cancer cells was reversed by mTORC1 inhibition, suggesting that elevated mTOR signaling downstream of LKB1 drives the metabolic phenotype of these cancers. We also observed hallmarks of the Warburg effect, notably increased intratumor glucose and lactate levels, in primary LKB1-deficient ErbB2-postive tumors, suggesting that LKB1 regulates glucose metabolism in tumors in situ. This is consistent with previous work showing enhanced glucose uptake by fluorodeoxyglucose (18F) positron emission tomography in benign LKB1
+/− colon polyps . Metabolic analysis also revealed that LKB1 loss promotes an increased bioenergetic state in ErbB2-positive tumors; the level of energy storage metabolites, particularly ATP and creatine, were elevated in LKB1-null tumors. Thus, silencing LKB1 may prime breast cancer cells for growth by modulating pro-growth glycolytic metabolism and enhancing ATP production and/or storage.
Given the role of LKB1 as a regulator of several protein kinase pathways, its loss likely affects multiple biological pathways in tumors in addition to metabolism. Epithelial integrity is an important parameter for tissue homeostasis, and loss of epithelial integrity as well as disruption of normal cellular polarization is often a precursor to metastasis [5, 29]. Our data suggest that loss of LKB1 leads to altered cell junction formation, reduced expression of epithelial markers, and increased migratory and invasive properties of breast cancer cells in vitro. It is unclear whether the metabolic changes induced by LKB1 loss contribute significantly to these phenotypes.
Clinical data shows LKB1 loss in more invasive cancers and in vitro data suggests association between loss of LKB1 and acquisition of pro-migratory and pro-invasive properties [46–48], However, despite these pro-growth and pro-metastatic phenotypes, we consistently observed a decrease in the ability of LKB1-deficient breast cancer cells to grow as metastases in the lung (Figure 3), suggesting that LKB1 is required for efficient tumor cell growth in a metastatic setting. The diminished lung metastatic burden in animals with LKB1-null ErbB2 breast tumors raises interesting questions regarding the fitness of LKB1-deficient tumor cells. The metastatic process represents a major energetic stress for the cells as they leave their native environment, travel through the blood, and ultimately seed in a new organ, where they must adapt to a new environment. Recent evidence suggests that LKB1 may be required for primary tumors to adapt to and survive metabolic stress. Models of mutant K-ras-driven lung tumorigenesis demonstrate that LKB1 loss accelerates lung tumor formation , but these tumors display increased sensitivity to apoptosis induced by the metabolic stressor phenformin . Consistent with these observations, we find that LKB1-deficient breast cancer cells are increasingly sensitive to glucose limitation. Likewise, LKB1-null NIC cells are unable to adapt their metabolism when challenged with the mitochondrial inhibitor metformin. The mechanisms that underlie this increased sensitivity to metabolic stress are still being examined.
One interesting aspect of the data presented here is that LKB1-deficient breast tumors display heightened energetics at the primary site, but appear to require LKB1 for efficient metastasis to the lung. It is possible that loss of LKB1 locks these cells into a specific mode of pro-growth metabolism, making them less able to adapt to changing tumor or metastatic microenvironments with fluctuating nutrient supply. ErbB2-positive breast tumor cells lacking LKB1 appear to exist in a pro-growth state of growth (that is, elevated Akt/mTOR, increased glycolysis); as demonstrated in Figure 7, these cells continue to maintain a pro-growth state even in the face of reduced nutrient availability. This may explain why dampening the pro-growth state of LKB1-null breast tumor cells, with rapamycin or similar agents, enhances their survival under low glucose conditions. Thus, despite its anti-proliferative effects, LKB1 may confer metabolic flexibility to tumor cells as they colonize and attempt to re-initiate growth in a foreign microenvironment. Collectively our data suggest that in breast cancer LKB1 represents a molecular switch that can regulate breast tumor growth in a stage-dependent manner; loss of LKB1 promotes oncogene-dependent tumorigenesis and early-stage growth in the primary site, but attenuates the growth of breast cancer cells as lung metastases.