Modulation of Mutant KrasG12D -Driven Lung Tumorigenesis In Vivo by Gain or Loss of PCDH7 Function

Abstract

PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. Although PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types. Given that PCDH7 is a potentially targetable molecule on the surface of cancer cells, further investigation of its role in tumorigenesis in vivo is needed to evaluate the therapeutic potential of its inhibition. Here, we report the analysis of novel PCDH7 gain- and loss-of-function mouse models and provide compelling evidence that this cell-surface protein acts as a potent lung cancer driver. Employing a Cre-inducible transgenic allele, we demonstrated that enforced PCDH7 expression significantly accelerates Kras^G12D -driven lung tumorigenesis and potentiates MAPK pathway activation. Furthermore, we performed in vivo somatic genome editing with CRISPR/Cas9 in Kras^LSL-G12D ; Tp53^fl/fl (KP) mice to assess the consequences of PCDH7 loss of function. Inactivation of PCDH7 in KP mice significantly reduced lung tumor development, prolonged survival, and diminished phospho-activation of ERK1/2. Together, these findings establish a critical oncogenic function for PCDH7 in vivo and highlight the therapeutic potential of PCDH7 inhibition for lung cancer. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the new inducible transgenic model described here provides a robust experimental system for broadly elucidating the effects of PCDH7 overexpression in vivo. IMPLICATIONS: In this study, we establish a critical oncogenic function for PCDH7 in vivo using novel mouse models and CRISPR/Cas9 genome editing, and we validate the therapeutic potential of PCDH7 inhibition for lung cancer.

Figure 1.. PCDH7 accelerates lung tumorigenesis in the Kras^LSL-G12D model.

A, Schematic depicting the PCDH7^LSL and Kras ^LSL-G12D transgenes and overview of experimental design. To induce lung tumors, Kras^LSL-G12D; PCDH7^LSL/LSL or control Kras^LSL-G12D mice were infected with Adeno-Cre through intratracheal administration. B, H&E staining of lung lobes harvested at 16 and 20 weeks post-infection. Five independent lobes from one animal of each genotype are shown. C, Tumor burden analysis at 16 and 20 weeks post-infection. n=5–6 mice/group, 5 sections/mouse analyzed. Tumors were counted and classified into atypical adenomatous hyperplasia (AAH)/bronchiolar hyperplasia (BH), adenomas, or adenocarcinomas. D, Weight of lungs collected from Kras^LSL-G12D; PCDH7^LSL/LSL or Kras^LSL-G12D mice at 20 weeks post-infection. E, IHC staining of Ki67 at 20 weeks and F, quantification of Ki67 index for lung sections harvested from Kras^LSL-G12D; PCDH7^LSL/LSL or control Kras^LSL-G12D mice at 16 and 20 weeks post-infection. n=2–4 mice/group, up to 10 fields quantified/animal. G, IHC staining of pERK1/2 for lung sections from Kras^LSL-G12D; PCDH7^LSL/LSL or control Kras^LSL-G12D mice at 16 weeks post-infection. H, quantification of p-ERK1/2 IHC staining at 16 and 20 weeks post-infection. n=2 mice/group, 7 fields quantified/animal.

Figure 2.. In vivo inactivation of Pcdh7 reduces lung tumor burden and prolongs survival of Kras^LSL-G12D; Tp53^fl/fl mice.

A, Schematic of in vivo CRISPR/Cas9 editing. KP mice were infected with a sg-control or sg-Pcdh7 through intratracheal administration of pSECC lentivirus expressing Cas9 and Cre. B, Survival analysis of KP mice infected with sg-control or sg-Pcdh7 lentivirus. C, H&E staining of lung lobes harvested at 10 weeks post-infection. D, Tumor burden analysis at 10 weeks post-infection. Tumors were counted and classified into AAH, BH, or adenomas based on histopathologic analysis. n=5–7 animals/group, 5 lobes/animal, 1 section/lobe analyzed. E, Total tumor area (inches²) at 10 weeks post-infection based on histopathologic analysis of lung sections using the NIH Image J program. F, Percent tumor area compared to total area of lung lobes at 10 weeks post-infection (determined using NIH Image J). G, H&E staining of lung lobes harvested at 20 weeks post-infection. H, Total tumor area (inches²) at 20 weeks post-infection based on histopathologic analysis of lung sections. I, Percent tumor area compared to total area of lung lobes at 20 weeks post-infection.

Figure 3.. Somatic knockout of Pcdh7 inhibits proliferation and reduces MAPK signaling in Kras^LSL-G12D; Tp53^fl/fl mice.

A, Western blot analysis of PCDH7, pERK1/2, and total ERK in normal lung tissues from uninfected KP mice, and lung tumors from KP mice infected with sg-control or sg-Pcdh7 lentivirus (20 weeks post-infection). B-C, Left, Ki67 immunohistochemistry (IHC) staining of lung sections harvested from KP mice infected with sg-control or sg-Pcdh7 lentivirus at 10 weeks (B) or 20 weeks (C) post-infection. Right, Ki67 index = percent of cells with a Ki67 positive signal / total cell number in each field. n=3 animals/group. D, pERK1/2 IHC staining of lung lobes from Kras^LSL-G12D; Tp53 fl/fl mice infected with sg-control or sg-Pcdh7 lentivirus at 10 weeks post-infection. E, Quantification of p-ERK1/2 IHC staining (represented by percent of p-ERK1/2 positive cells) at 10 and 20 weeks post-infection. n=2–3 animals/group, 6–10 fields quantified/animal. F, TUNEL staining of lung lobes from Kras^LSL-G12D; Tp53 fl/fl mice infected with sg-control or sg-Pcdh7 lentivirus at 10 weeks post-infection. G, Quantification of TUNEL index (represented by percent of TUNEL positive cells) at 10 and 20 weeks post-infection. n=2–3 animals/group, 15 independent fields quantified/animal.

Figure 4.. PCDH7 modulates expression of PP2A targets pERK1/2 and pRB.

A, Western blot analysis of PCDH7, pERK, total ERK, pRb and total RB levels in HBECs expressing KRAS^G12V, PCDH7, or both. B, PCDH7 inhibition with siRNAs in HBEC-shp53-KRAS^G12V-PCDH7 cells and its effects on pERK and pRb signaling, as indicated by western blot analysis. C, Western blot analysis of PCDH7, pRB, and total RB expression in lung tumors harvested from KP mice with sg-control or sg-Pcdh7 lentivirus at 20 weeks post-infection. D, Quantification of pRB protein levels shown in (C). E, Western blot analysis showing diminished pERK1/2 and pRB protein in human NSCLC H1944 xenografts with PCDH7 sgRNA.