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

Abstract

Background CRIPTO 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. Methods We 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. Results ALK4L75A-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. Conclusions Cancer 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.

Whence CRIPTO: The Reemergence of an Oncofetal Factor in ‘Wounds’ That Fail to Heal

“…Should these populations represent de-differentiated cell types, the obvious question is what factors reprogram the system to enable CRIPTO reemergence? Recently, we (BTS) showed within MDA-MB-231 xenograft tumors that CRIPTO immunoreactivity localizes to tumor microdomains exhibiting hallmarks of stress such as hypoxia [ 118 ]. Although it is known that such tumor microdomains include both xenografted tumor cells and infiltrating host stroma, it is not yet clear which cells are signaling to which other cells through the agency of CRIPTO.…”

“…This harkens to the integration of hypoxic stress and CRIPTO signaling in HSCs, ischemia models and cardiomyocytes [ 39 , 83 , 84 ]. Alternatively, these zones are likely starved for other nutrients and, in this regard, we found that nutrient deprivation in vitro (i.e., serum withdrawal or glycolytic blockade) confers cancer cell CRIPTO dependency [ 118 ]. Thus, microenvironmental cues such as hypoxia or metabolic restriction may explain the spatial heterogeneity of CRIPTO expression tumors.…”

“…A fraction of GRP78 localizes to the cell surface from the endoplasmic reticulum in the presence of diverse stresses where it binds CRIPTO and facilitates downstream signaling throug c-Src and NODAL [ 100 , 119 ]. Supporting the importance of contextual alterations to the surface proteome, GRP78 and CRIPTO co-localize along acellular regions within xenograft mammary tumors [ 118 ]. These acellular regions are characterized by relatively high levels of hypoxia.…”

“…Consistent with this, in cancer, we have shown inhibition of CRIPTO with a soluble inhibitor, Alk4 L75A -Fc, reduces primary and metastatic tumor burden in a breast cancer xenograft model. This occurs despite the fact Alk4 L75A -Fc did not appreciably disrupt CRIPTO-NODAL signaling in cis [ 118 ].…”

“…As previously discussed, CRIPTO overexpression alone in the mammary gland is not sufficient to drive tumorigenesis [ 104 ]. However, CRIPTO signaling blockade in MDA-MB-231 mammary tumor xenografts attenuates primary tumor burden, although inhibition of CRIPTO is not sufficient to cause regression [ 118 ]. Similar effects were observed in a metastatic mouse model using CRISPR-Cas9 mediated knockout of CRIPTO in the TNBC-like JygMC(A) cell line [ 101 ].…”