Suppression of lung adenocarcinoma progression by Nkx2-1

Abstract

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Figure 1. A lentiviral vector-induced mouse model of lung adenocarcinoma identifies gene expression alterations during tumour progression

a, Infection of Kras^LSL-G12D/+;p53^flox/flox mice with Cre-expressing lentiviral vectors initiates lung adenocarcinoma. b, Linker-mediated PCR cloning of the lentiviral integration site in metastases (Met) allows specific PCR amplification of that lentiviral-integration (lower band) to identify which primary tumour gave rise to the metastases. Top band is a control product. c, Southern blot on cell lines for the integrated lentiviral genome. d, Representative images of livers after intrasplenic transplantation of T_nonMet or T_Met cells. Scale bar = 0.5cm. e, Quantification of liver nodules after intrasplenic injection of two T_nonMet and T_Met cell lines. f, Gene expression alterations (log₂) between T_nonMet and T_Met/Met samples.

Figure 2. Reduced Nkx2-1 in advanced lung adenocarcinoma correlates with a less differentiated state

a, Cross-species analysis of human lung adenocarcinoma patient outcome (likelihood ratio with the sign from correlation value) versus differential gene expression in murine T_nonMet cells. b, Nkx2-1 protein is absent from T_Met and Met-derived cell lines. c–d, Nkx2-1 expression is high in well differentiated adenomas and early murine adenocarcinoma (top) but is downregulated in moderately to poorly differentiated advanced carcinomas (bottom). Scale bar = 50μm. Upper inlay Nkx2-1 staining. Lower inlay H+E staining. e, Quantification of Nkx2-1 expression in murine lung tumours relative to tumour grade from most differentiated (atypical adenomous hyperplasia (AAH)) to least differentiated (Poor)).

Figure 3. Nkx2-1 controls lung adenocarcinoma differentiation and restricts metastatic ability

a, Nkx2-1 protein expression in T_Met cells. b, Nkx2-1 expression reduces lung nodule formation after intravenous transplantation. p < 0.002. c, Quantification of Nkx2-1 in lung nodules after T_Met or T_Met-Nkx2-1 transplantation. n=3/group. d, Association of Nkx2-1 expression with differentiation state after T_Met or T_Met-Nkx2-1 transplantation. Fisher’s exact test on the association of differentiation state with Nkx2-1 p < 0.002. e, Nkx2-1 expression reduces anchorage-independent growth of T_Met cells. Representative images and colony number (mean +/− SD of quadruplicate wells, p < 0.0001). f, Nkx2-1 knockdown increases liver nodules after intrasplenic injection (top) and lung nodules after intravenous transplantation (middle) of T_nonMet cells. Representative of 7 mice/group. shNkx2-1 enhanced anchorage-independent growth of T_nonMet cells (bottom). Representative images and colony number (mean +/− SD of triplicate wells, p < 0.0001). g. Induction of tumours in Kras^LSL-G12D/+;p53^flox/flox mice with Nkx2-1/Cre lentivirus reduces the development of advanced tumours (grades 3&4). Numbers indicate percent of tumours in each group.

Figure 4. Nkx2-1 regulates the expression of Hmga2 in advanced lung adenocarcinoma

a, Nkx2-1 knockdown derepresses Hmga2 in T_nonMet cell lines. b, Hmga2 and Nkx2-1 are reciprocally expressed in Kras^G12D/+;p53^Δ/Δ murine lung adenocarcinomas. Scale bar = 50μm. Inlaid images show cellular features and protein localization. c, Hmga2 and Nkx2-1 expression in advanced Kras^G12D/+;p53^Δ/Δ murine lung adenocarcinomas. Fisher’s exact test, p-value < 10⁻¹¹. d, Early Kras^G12D/+;p53^Δ/Δ tumours are Nkx2-1^posHmga2^neg. e, NKX2-1 and HMGA2 expression in human lung adenocarcinomas. Large numbers are percentages. Small numbers are absolute numbers. f, Hmga2 knockdown reduces the tumourigenic potential of T_nonMet-shNkx2-1 cells after intravenous transplantation. Control samples include the parental T_nonMet-shNkx2-1 cells (grey circle) and cells infected by a control retrovirus (black circles). p < 0.003. g, shHmga2 reduces anchorage-independent growth of a metastasis-derived cell line (Met). Representative images and colony number (mean +/− SD of quadruplicate wells, p < 0.0001). h, shHmga2 reduces the tumour-seeding potential of a Met cell line after intravenous transplantation. p <0.0001.