No Fib, Nfib is novel small cell lung cancer oncogene

Science Spotlight

No Fib, Nfib is novel small cell lung cancer oncogene

From the MacPherson Lab (Human Biology Division)

Nov. 21, 2016
Chart showing NFIB expression decreases the age of onset for SCLC model

NFIB expression increases age of onset and mortality of small cell lung cancer in vivo. Kaplan-Meier survival curve for transgenic model with inducible NFIB expression that histologically exhibits small cell lung cancer phenotypes and neuroendocrine expression profile (CGRP staining).

Image provided by Dr. David MacPherson

In an effort to develop patient-specific precision therapies, scientists are gathering enormous amounts of data concerning the molecular features of tumors. When it comes to classifying cancers, the broadest level is the primary site of a tumor and its pathology (such ductal versus lobular breast cancer). Additionally, tumors can be classified and grouped by somatic mutations, RNA profiles, copy number alterations, and protein expression levels. This approach has been very successful in understanding many tumor types, and recently it has been adopted for understanding how tumors adapt and evolve. Clinicians are using molecular features to compare tumor samples before and after recurrence in patients to understand how cancers respond and adapt to conventional therapies. Laboratory scientists are using these approaches in cancer models to compare tumors from a primary site and their metastatic counterparts to understand what changes enable a metastatic population to expand, disseminate, and implant elsewhere. Researchers in the MacPherson Lab (Human Biology Division) took this approach to understand how small cell lung cancer (SCLC) initiates and undergoes progression to metastasize to the liver. Fred Hutch postdoc, Dr. Nan Wu and colleagues identified a novel oncogene that stimulated both metastatic and primary SCLC. These findings were recently published in Oncotarget.

For this study, researchers used a transgenic mouse model of small cell lung cancer. In this model the tumor suppressor genes p53 and Rb have critical exons flanked by loxP sites. Thus these genes can be deleted in any cells exposed to CRE recombinase. To delete these genes specifically in the lungs, adenovirus containing CRE driven by a tissue-specific promoter is delivered directly through the trachea. This knock out combination induces primary tumors in the lung and metastatic lesions within the liver. Researchers isolated each tumor type and performed various molecular profiling technologies. When comparing loss or gain of chromosome regions researchers identified a region on chromosome 4 that was sometimes amplified in primary tumors but much more frequently amplified in metastatic tumors. This region of chromosome 4 contains the gene Nfib and when amplified, an increase in Nfib transcripts was also observed. Like many oncogenes Nfib encodes a transcriptional regulator that is essential for embryonic lung and brain development. It has been shown to increase tumor cell proliferation, but previously had not been evaluated in vivo for its function as an oncogene.

To understand the role of Nfib in SCLC tumorigenesis researchers created a new transgenic model. Starting with the same CRE-inducible knock out of p53 and Rb, they introduced a tetracycline-inducible version of the Nfib gene. Thus the addition of CRE deletes tumor suppressor genes and addition of doxycycline induces high levels of Nfib expression. This genetic combination accelerated SCLC, decreasing age of onset over p53 and Rb deletion alone, showing that NFIB functions as an oncogene. However, in this model the frequency of metastasis in p53/Rb SCLC was already high and metastatic tumor burden did not increase with Nfib overexpression.

 

Immunoblot and graph showing CRISPR/Cas9 deletion of NFIB is potent and decreases tumor growth

CRISPR/Cas9 mediated deletion of NFIB in small cell lung cancer cell line is robust (left) and deletion decreases cell proliferation (right).

Image provided by Dr. David MacPherson

Cell lines were isolated from the mouse model for further testing. The p53/Rb-null cells were continually cultured in the presence of doxycycline to maintain high levels of NFIB expression, and in fact when doxycycline was removed cell proliferation decreased dramatically. The decrease in proliferation coincided both with increased cell death/apoptosis (caspase activity) and cell cycle arrest (cyclin dependent kinase inhibition). These results suggest many SCLC cells become dependent on NFIB activities.  To test this hypothesis, the researchers deleted Nfib in a human SCLC cell line using CRISPR. NFIB loss decreased proliferation in these cells and reduced their migratory behavior in vitro.

To begin understanding what pathways NFIB was activating or repressing researchers compared RNA expression profiles for three different data sets: (1) Liver metastases from p53, Rb mice, comparing high and low Nfibexpressing samples; (2) Primary tumors from p53, Rb, Nfib mice, comparing with and without doxycycline; and (3) tumor cells from p53, Rb, Nfib mice, comparing with and without doxycycline. From these three comparisons 66 genes were identified as having altered levels due to Nfib expression. Interestingly, when the three data sets were compared, the highest ranked genes with increased expression were those involved in axon growth and guidance. This finding is consistent with a more migratory, metastatic population.

Further characterizing the mechanism by which NFIB stimulates tumor growth and metastasis is a high priority, as Dr. David MacPherson explained, “We are now interested in identifying NFIB target genes that mediate oncogenic effects of Nfib overexpression.  We have identified a panel of candidate genes that are consistently and strongly regulated by NFIB that we are perturbing to test for oncogenic activity.” Another future route of research is to understand the temporal role of NFIB, “We have also initiated in vivo experiments to test the importance of NFIB for maintenance of primary and metastatic SCLC, as a nice feature of the new mouse model is we can remove doxycycline from the mouse diet and turn off Nfib in established tumors.” said Dr. MacPherson.

Good scientific findings often raise more questions than they answer. Understanding the specific activity of NFIB in lung and other cancers may yield novel therapeutic targets and an increased understanding of transcriptional regulation.


Wu N, Jia D, Ibrahim AH, Bachurski CJ, Gronostajski RM, MacPherson D. 2016. NFIB overexpression cooperates with Rb/p53 deletion to promote small cell lung cancer. Oncotarget. 
7(36):57514-24.

 

Funding for this research was provided by the Cancer Research Institute and NYSTEM.