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. 2013 Sep 25;8(9):e73449.
doi: 10.1371/journal.pone.0073449. eCollection 2013.

The LKB1 tumor suppressor as a biomarker in mouse and human tissues

Yuji Nakada  1 , Thomas G Stewart, Christopher G Peña, Song Zhang, Ni Zhao, Nabeel Bardeesy, Norman E Sharpless, Kwok-Kin Wong, D Neil Hayes, Diego H Castrillon
Affiliations

The LKB1 tumor suppressor as a biomarker in mouse and human tissues

Yuji Nakada et al. PLoS One. .

Abstract

Germline mutations in the LKB1 gene (also known as STK11) cause the Peutz-Jeghers Syndrome, and somatic loss of LKB1 has emerged as causal event in a wide range of human malignancies, including melanoma, lung cancer, and cervical cancer. The LKB1 protein is a serine-threonine kinase that phosphorylates AMP-activated protein kinase (AMPK) and other downstream targets. Conditional knockout studies in mouse models have consistently shown that LKB1 loss promotes a highly-metastatic phenotype in diverse tissues, and human studies have demonstrated a strong association between LKB1 inactivation and tumor recurrence. Furthermore, LKB1 deficiency confers sensitivity to distinct classes of anticancer drugs. The ability to reliably identify LKB1-deficient tumors is thus likely to have important prognostic and predictive implications. Previous research studies have employed polyclonal antibodies with limited success, and there is no widely-employed immunohistochemical assay for LKB1. Here we report an assay based on a rabbit monoclonal antibody that can reliably detect endogenous LKB1 protein (and its absence) in mouse and human formalin-fixed, paraffin-embedded tissues. LKB1 protein levels determined through this assay correlated strongly with AMPK phosphorylation both in mouse and human tumors, and with mRNA levels in human tumors. Our studies fully validate this immunohistochemical assay for LKB1 in paraffin-embedded formalin tissue sections. This assay should be broadly useful for research studies employing mouse models and also for the development of human tissue-based assays for LKB1 in diverse clinical settings.

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Conflict of interest statement

Competing Interests: DC, KW, NB and NS are inventors on an application (United States patent pending) of DNA-based methods to employ LKB1 status for diagnostic and prognostic clinical tests. This invention does not utilize the methods or antibody described in this manuscript. These investigators are paid consultants of Molecular MD (Portland, OR), which has a license agreement with these authors' respective academic institutions. NH is an inventor on an application (United States patent pending) for the use of LKB1 as a prognostic factor – this invention also does not utilize the methods or antibody described in this manuscript.

Figures

Figure 1
Figure 1. Validation of LKB1 rabbit monoclonal antibody D60C5 for immunohistochemistry in human and mouse paraffin-embedded, formalin-fixed samples.
A, Human cervical cancer cell lines. HeLa/Lkb1  =  HeLa cells following transduction of lentivirus harboring a human LKB1 cDNA inducible expression construct. Relative expression levels of Lkb1 protein are indicated in parentheses. B, Mouse tissues (endometrium and lung) from animals harboring floxed alleles of Lkb1 following Cre-mediated recombination with Sprr2f-Cre (endometrium) or nasal-instillation of Adeno-Cre virus (lung). Distinct Lkb1-null clones are indicated by dashed lines (endometrium) or arrows (lung). Bars = 10 µm for each panel. Asterisks in the lung panels show invasive cancer cells subjacent to the dysplastic epithelium; these invasive cancer cells are also clearly Lkb1-null. C, Percent of Lkb1-null cells in Sprr2f-Cre; Lkb1L /L female mice by immunohistochemistry at 3, 6, 12, and 20 weeks of age. Error bar  =  S.E.M. D, Normal patterns of LKB1 protein in human lung and oviduct highlighting localization to the apical surface of ciliated cells (arrows). Note: in the oviduct, ciliated epithelial cells (arrows) are interspersed among nonciliated cells. Bars = 10 µm in both panels.
Figure 2
Figure 2. Testing of another α-LKB1 monoclonal antibody (Ley 37D/G6).
Tissue sections are from the uterus of a 6-week old Sprr2f-Cre; Lkb1L /L female mouse. Rabbit monoclonal D60C5 readily distinguishes LKB1 positive from negative cells as shown previously. In contrast, the mouse monoclonal antibody Ley 37D/G6 shows a homogeneous pattern throughout the endometrial epithelium (serial step section) and fails to distinguish between LKB1 positive and negative cells. Size bars = 100 µ for each panel.
Figure 3
Figure 3. Validation of rabbit monoclonal antibody D60C5 by Western blotting.
Positions of molecular weight standards (kilodaltons) are shown to the left of each blot. A, HeLa cells harboring Tet-On construct inducible with doxycycline. B, Comprehensive uterine cancer cell line panel (endometrial and cervical). Note: C4I harbors biallelic mutations of LKB1: a chromosomal deletion plus a point mutation that does not affect protein levels . CaSki, C33, and ME180 do not harbor LKB1 mutations . Endo was derived from normal endocervical epithelium immortalized with HPV E6/E7 .
Figure 4
Figure 4. Scoring schema for LKB1 and pAMPKα (Thr172) expression in human lung cancer specimens.
Tissues were paraffin-embedded and fixed in formalin. Only staining in the malignant epithelial cells was scored. A, LKB1 immunohistochemistry and representative cases illustrating histologic scores. B, pAMPKα (Thr172) immunohistochemistry and representative cases illustrating histologic scores. The dynamic range was somewhat lower for pAMPKα (Thr172) vs. LKB1 but a wide range of staining intensities was also observed. Bar = 10 µm in all panels; all panels are at same magnification.
Figure 5
Figure 5. Validation of LKB1 antibody in situ assay in human lung tumor specimens.
A, LKB1 protein expression vs. gene expression scores. Researchers used the Agilent 44(p<0.01). B, Box plots showing comparison of gene expression scores in cases with confirmed LKB1 loss-of-function mutations vs. cases with no mutations. C, Protein expression by mutation status providing visual comparison of cases with mutation vs. no mutation. The x-axis shows the percentage of cases per LKB1 score (i.e. each side adds up to 1). The unsymmetrical shape indicates differences between the groups.
Figure 6
Figure 6. Correlation between LKB1 and pAMPKα (Thr172) scores.
Heat map shows associations between LKB1 and pAMPKα (Thr172) protein expression scores. Kendall's tau provides a summary of the correlation (τκ = 0.49, p<0.001).
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References

    1. Wingo SN, Gallardo TD, Akbay EA, Liang MC, Contreras CM, et al. (2009) Somatic LKB1 mutations promote cervical cancer progression. PLoS One 4: e5137. - PMC - PubMed
    1. Ji H, Ramsey MR, Hayes DN, Fan C, McNamara K, et al. (2007) LKB1 modulates lung cancer differentiation and metastasis. Nature 448: 807–810. - PubMed
    1. Shah U, Sharpless NE, Hayes DN (2008) LKB1 and lung cancer: more than the usual suspects. Cancer Res 68: 3562–3565. - PubMed
    1. Guldberg P, thor Straten P, Ahrenkiel V, Seremet T, Kirkin AF, et al. (1999) Somatic mutation of the Peutz-Jeghers syndrome gene, LKB1/STK11, in malignant melanoma. Oncogene 18: 1777–1780. - PubMed
    1. Liu W, Monahan KB, Pfefferle AD, Shimamura T, Sorrentino J, et al. (2012) LKB1/STK11 inactivation leads to expansion of a prometastatic tumor subpopulation in melanoma. Cancer Cell 21: 751–764. - PMC - PubMed

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