CRIPTO antagonist ALK4L75A-Fc inhibits breast cancer cell plasticity and adaptation to stress

Ozlen Balcioglu,Kishan Bhakta,Supraja Ranganathan,Brooke L Gates,Richard E Heinz,Evan Booker,David W Freeman,Hyrum T Diesen,Mathias Leblanc,Berhane M Hagos,Masami Kachi,Peter C Gray,Benjamin T Spike,Elnaz Mirzaei Mehrabad

doi:10.1186/s13058-020-01361-z

Abstract

BackgroundCRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells.MethodsWe focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach.ResultsALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden.ConclusionsCancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Highlights

CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis
We show that treatment of NCCIT cells with ALK4L75A-Fc inhibits NCCIT cell growth (Fig. 1a) and that this effect is rescued by addition of soluble recombinant CRIPTO protein (Fig. 1b)
We show that the CRIPTO directed antagonist ALK4L75A-Fc is active against poorly differentiated triple negative breast cancer (TNBC) cells in vitro and in vivo, and that it blocks their ability to adapt to stresses such as nutrient

Summary

Introduction

CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. CRIPTO is a small GPI-anchored signaling protein that regulates normal stem cell activity and oncogenesis [5,6,7,8]. It is selectively upregulated in multiple human tumor types including ~ 80% of human breast cancers where it is associated with poor patient prognosis [9,10,11]. CRIPTO knockdown inhibits proliferation of human MDA-MB-468 and SKBR3 breast cancer cell lines in vitro [18], while CRIPTO knockdown in a triple negative breast cancer (TNBC)-like transplantable mouse mammary tumor model blocks progression [19]. Several groups are seeking to develop CRIPTO as a biomarker of tumor aggressiveness and validate therapeutic modalities directed at blocking CRIPTO-induced oncogenesis [19, 23,24,25]

Methods

Results

Discussion

Conclusion