RNAi screen identifies essential regulators of human brain metastasis-initiating cells

Abstract

Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

Therapeutic Targeting of the Premetastatic Stage in Human Lung-to-Brain Metastasis

“…We utilized early-passage BM cell lines derived from primary patient samples of lung-to-BM in our work, as these samples are enriched for BMICs that have already successfully completed the metastatic process. Previous work in our lab successfully established preclinical models of lung-to-brain BM (14,31). Briefly, we injected mice through 3 different injection routes: (i) intracranial (ICr), (ii) intrathoracic injections (IT), and (iii) intracardiac injections (ICa), where we were able to replicate the premetastatic and macrometastatic stages from IT and ICa injections respectively (14).…”

“…BMs were processed and maintained in tumor sphere media (TSM) as previously described (14,15). BMICs were grown as tumorspheres that were maintained at 37 C with a humidified atmosphere of 5% CO 2 .…”

“…Injections were performed as previously described for intracranial (ICr), intrathoracic (IT), and intracardiac (ICr) routes (Supplementary Table S1A; ref. 14).…”

Novel therapeutic targets for cancer metastasis

“…Many of the putative drug targets for metastasis have no known function or enzymatic activity and in silico analysis often finds these genes categorized into seemingly unrelated pathways such as lipid metabolism or vesicular transport [94][95][96][97].…”

Hirnmetastasen

“…In einer prospektiven Studie bildeten nur Bronchialkarzinome Hirnmetastasen, wenn SPOCK1 in den Lungenkrebs-Biopsien überexprimiert war (2). Außerdem fand die ihre Arbeitsgruppe, dass eine hohe Expression von humanen Leukozyten-Antigen(HLA)-G-Molekülen mit einem geringeren Überleben bei Patienten mit Bronchial-und Mammakarzinom sowie Melanomen assoziiert waren.…”