Friday, June 22, 2012

LKB1 and Melanoma

LKB1 has been demonstrated to be the underlying control element in Peutz-Jeghers syndrome, a proliferative melanocytic genetically dominant disorder. It controls certain pathways and as a result can be considered as a candidate in the development and progression of melanoma. Generally LKB1 is a gene whose protein stabilizes the growth and location of melanocytes. Understanding its impact in Peutz-Jeghers allows one to examine what happens when its function is suppressed in melanoma. Albeit not an initiator in the process, its aberration in a melanocyte argues for movement and loss of control.
In a recent paper by Liu et al the authors examine this premise and conclude that loss of LKB1 is significant especially in metastatic evolution. As Liu et al state:

Germline mutations in LKB1 (STK11) are associated with the Peutz-Jeghers syndrome (PJS), which includes aberrant mucocutaneous pigmentation, and somatic LKB1 mutations occur in 10% of cutaneous melanoma. By somatically inactivating Lkb1 with K-Ras activation (±p53 loss) in murine melanocytes, we observed variably pigmented and highly metastatic melanoma with 100% penetrance. LKB1 deficiency resulted in increased phosphorylation of the SRC family kinase (SFK) YES, increased expression of WNT target genes, and expansion of a CD24+ cell population, which showed increased metastatic behavior in vitro and in vivo relative to isogenic CD24 cells. These results suggest that LKB1 inactivation in the context of RAS activation facilitates metastasis by inducing an SFK-dependent expansion of a prometastatic, CD24+ tumor subpopulation.

Earlier work by Zheng et al noted:

The LKB1-AMPK signaling pathway serves as a critical cellular sensor coupling energy homeostasis to cell growth, proliferation, and survival. However, how tumor cells suppress this signaling pathway to gain growth advantage under conditions of energy stress is largely unknown.

Here, we show that AMPK activation is suppressed in melanoma cells with the B-RAF V600E mutation and that downregulation of B-RAF signaling activates AMPK. We find that in these cells LKB1 is phosphorylated by ERK and Rsk, two kinases downstream of B-RAF, and that this phosphorylation compromises the ability of LKB1 to bind and activate AMPK. Furthermore, expression of a phosphorylation-deficient mutant of LKB1 allows activation of AMPK and inhibits melanoma cell proliferation and anchorage-independent cell growth.

Our findings provide a molecular linkage between the LKB1-AMPK and the RAF-MEK-ERK pathways and suggest that suppression of LKB1 function by B-RAF V600E plays an important role in B-RAF V600E-driven tumorigenesis.

Thus Zheng et al putatively identified these two pathways as sources for melanoma development. Liu et al appear to have extended this to metastasis.

Now in a paper by Bauer and Stratakis the authors provide an excellent overview of the controlling pathways. We provide a revised version of their pathway controls in a normal melanocyte below.

The LKB1 gene, also called STK11, which encodes a member of the serine/threonine kinase, regulates cell polarity and functions as a tumour suppressor. This is clearly demonstrated in the above.

Now Liu et al state regarding this pathway model:

Two independent pathways appear to be critically important in regulating cell growth in response to nutrient supply and mitogenic stimulation:

(i) the PKA/PRKAR1A-LKB1 tumour suppressor protein pathway, acting via AMPK, and

(ii) the PI3K/AKT pathway.

Recent evidence suggests that the tumour suppressor gene complex, TSC1/TSC2, orchestrates the signal from both pathways to the downstream target, mTOR, which in turn regulates the ribosomal protein S6 and 4EBP-1, a repressor of the translational initiation factor eIF4E. In this model, at times of nutrient stress LKB1/AMPK activation of the TSC1/TSC2 complex results in inhibition of mTOR and a decrease in protein synthesis. Under stimulation of mitogenic pathways, PI3K phosphorylates PIP2 to PIP3 resulting in recruitment of AKT to the membrane where it is activated by PDK1. Activated AKT inhibits the TSC1/TSC2 tumour suppressor complex leading to increased mTOR activity. In the later pathway, PTEN antagonises PIP3 action through dephosphorylation, and thus provides an ‘‘off’’ switch for regulating mitogenic pathway induced cellular growth and proliferation.

Cross talk of several other pathways appears to play important regulatory roles in the lentiginoses syndromes to include the Ras/MAPK pathway in the regulation of translation, the LKB1 pathway in cellular polarity, the AKT pathway (as well as the TSC1/TSC2 complex) in the regulation of the Wnt/GSK3b/b-Cat pathway, and the BMP pathway in the regulation of PTEN (see text for further discussion). Lastly, both PTEN and mTOR appear to have negative regulatory effects on VEGF through loss of stabilisation of the hypoxia inducible transcription factor 1 (HIF1).

When LKB1 is inactivated we have the following changes observed:

These models or Bauer and Stratakis are compelling and establish a paradigm which the work of Liu et al can be considered.

Let us go back to LKB1 and its function. From NLM database we have[1]:

LKB1 is a primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK), a necessary element in cell metabolism that is required for maintaining energy homeostasis. It is now clear that LKB1 exerts its growth suppressing effects by activating a group of other ~14 kinases, comprising AMPK and AMPK-related kinases.

Activation of AMPK by LKB1 suppresses growth and proliferation when energy and nutrient levels are scarce. Activation of AMPK-related kinases by LKB1 plays vital roles maintaining cell polarity thereby inhibiting inappropriate expansion of tumour cells. A picture from current research is emerging that loss of LKB1 leads to disorganization of cell polarity and facilitates tumour growth under energetically unfavorable conditions. Also it is known as PJS; LKB1; hLKB1.

This gene, which encodes a member of the serine/threonine kinase family, regulates cell polarity and functions as a tumor suppressor. Mutations in this gene have been associated with Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by the growth of polyps in the gastrointestinal tract, pigmented macules on the skin and mouth, and other neoplasms. Alternate transcriptional splice variants of this gene have been observed but have not been thoroughly characterized.

From the results of Shaw et al we have[2]:

AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status found in all eukaryotic cells. AMPK is activated by stimuli that increase the cellular AMP/ATP ratio. Essential to activation of AMPK is its phosphorylation at Thr-172 by an upstream kinase, AMPKK, whose identity in mammalian cells has remained elusive.

Here we present biochemical and genetic evidence indicating that the LKB1 serine/threonine kinase, the gene inactivated in the Peutz-Jeghers familial cancer syndrome, is the dominant regulator of AMPK activation in several mammalian cell types. We show that LKB1 directly phosphorylates Thr-172 of AMPKalpha in vitro and activates its kinase activity.

LKB1-deficient murine embryonic fibroblasts show nearly complete loss of Thr-172 phosphorylation and downstream AMPK signaling in response to a variety of stimuli that activate AMPK. Reintroduction of WT, but not kinase-dead, LKB1 into these cells restores AMPK activity. Furthermore, we show that LKB1 plays a biologically significant role in this pathway, because LKB1-deficient cells are hypersensitive to apoptosis induced by energy stress.

On the basis of these results, we propose a model to explain the apparent paradox that LKB1 is a tumor suppressor, yet cells lacking LKB1 are resistant to cell transformation by conventional oncogenes and are sensitive to killing in response to agents that elevate AMP. The role of LKB1/AMPK in the survival of a subset of genetically defined tumor cells may provide opportunities for cancer therapeutics.
Also Shaw et al demonstrate several ways in which LKB1 can function when activated in vivo from either a basal or non-basal state. The description can be shown in the following Figure:
Shaw et al describe the above as follows:

Model for LKB1 as a sensor of low energy and negative regulator of tumorigenesis and apoptosis. Under basal conditions, LKB1 serves as a sensor of low energy, keeping ATP-consuming processes including protein synthesis in check via AMPK phosphorylation of TSC2.

In response to stresses such as low glucose, hypoxia, nutrient deprivation, or mitochondrial poisons, LKB1 phosphorylates AMPK, which shuts off ATP-consuming processes and up-regulates ATP production to offset the elevated AMP/ATP ratio. This activity prevents the cells from going into apoptosis in response to elevated AMP. In LKB1-deficient cells, under some basal conditions, there may be increases in TOR signaling due to the lack of TSC2 phosphorylation by AMPK, resulting in increased growth or tumorigenic potential. In response to further increases in intracellular AMP, these cells have no mechanism to offset the elevated AMP and go straight into apoptosis.

However, although this is an interesting and compelling description of the metastatic driving factors, there are a multiple set of issues still outstanding:

1. Metastatic behavior implies the ability of the malignant melanocyte to migrate at will within the body. Movement of the melanocyte requires breaking of the E cadherin bonds with the adjacent keratinocytes. Thus is there a sequence of genetic changes and how does this putative mechanism relate to that of the E cadherin mechanism.

As Baas et al state:

A second prominent aspect of polarized simple epithelia is the presence of junctional complexes at the apical boundaries between neighboring cells. These junctions form an impenetrable seal between cells and provide strength to the epithelial sheet by serving as anchoring sites for cytoskeletal elements including the brush border.

We found that LS174T cells do not express junctional proteins, such as ZO-1, and are homozygous mutant for E-cadherin. By contrast, DLD-1 cells are capable of forming tight junctions and adhesion junctions when grown to confluency and appear to express most junctional compo­nents already at low-cell density.

We determined the localization of the tight junction component ZO-1 and of the adherens junction protein p120 before and after activation of LKB1 in DLD-1-W5 cells grown at very low density.

2. LKB1 is a gene related to the control from decreased nutrients. However we have the angiogenesis issue related to the increased nutrition of malignant cells. However on the counter side we have the Warburg effect as a counter to normal metabolism, namely cancer cells are anaerobic metabolic systems. What is the balance between the two?

3. Is the LKB1 mutation one of random gene mutations or is it a direct consequence of other downstream mutations? Is perhaps this loss of LKB1 a result of some induced miRNA effect in vivo?

This is an interesting result and very much worth following.


1.               Baas, A., et al, Complete Polarization of Single Intestinal Epithelial Cells upon Activation of LKB1 by STRAD, Cell, Vol. 116, 457–466, February 6, 2004.
2.               Boudeau J., et al, Analysis of the LKB1-STRAD-MO25 complex, Journal of Cell Science 117, 6365-6375 Published by The Company of Biologists 2004.
3.               Shaw, R.,  et al, The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress, Proc Natl Acad Sci U S A. 2004 Mar 9;101(10):3329-35. Epub 2004 Feb 25.
4.               Suh, B., B. Hille, PIP2 is a necessary cofactor for ion channel function: How and why? Annu Rev Biophys. 2008; 37: 175–195.
5.               Tiainen, M. et al, Growth arrest by the LKB1 tumor suppressor: induction of p21 WAF1/CIP1 , Human Molecular Genetics, 2002, Vol. 11, No. 13 1497–1504.
6.               Trojan, J., et al, 5'-CpG island methylation of the LKB1/STK11 promoter and allelic loss at chromosome 19p13.3 in sporadic colorectal cancer, Jrl Med Gen 2000;47:272–276.
7.               Wang J., et al, Germline mutations of the LKB1 (STK11) gene in Peutz-Jeghers patients, J Med Genet 1999;36:365–368.
8.               Zheng, B., et al, Oncogenic B-RAF Negatively Regulates the Tumor Suppressor LKB1 to Promote Melanoma Cell Proliferation, Molecular Cell 33, 237–247, January 30, 2009.
9.               Zigler, M., et al, PAR-1 and thrombin: the ties that bind the microenvironment to melanoma metastasis, Cancer Res. 2011 Nov 1; 71(21):6561-6. Epub 2011 Oct 18.