Trop-2, Na+/K+ ATPase, CD9, PKCa, cofilin assemble a membrane signaling super-complex that drives colorectal cancer growth and invasion

Trop-2, Na+/K+ ATPase, CD9, PKCa, cofilin assemble a membrane signaling super-complex that drives colorectal cancer growth and invasion

Trop-2 is a dimeric transmembrane signal transducer expressed by normal epithelia at various stages of differentiation and strongly up-regulated in several human carcinomas.

We have previously demonstrated that Trop-2 is a critical driver of tumor growth and metastatic dissemination, making this molecule an ideal candidate target for anticancer therapy. Indeed, very much interest has been recently focused on developing Antibody-Drug-Conjugates able to specifically bind Trop-2 in cancer. The most recent example of these new therapeutic agents is the humanized anti-Trop-2 antibody–SN-38 drug conjugate Sacituzumab govitecan-hziy (Trodelvy), which was approved by the Food and Drug Administration (FDA) for treatment of patients with metastatic triple-negative breast cancers and urothelial cancers.

In this study we defined the membrane-to-nucleus signaling network driven by Trop-2 to induce colorectal cancer cell growth. Various signaling tiles of this complex puzzle were previously discovered by us and others, however the connections between these piece were much less defined.

Analysis by proteomics/tandem mass spectrometry as well as by advanced fluorescence microscopy (e.g. genetically encoded Ca2+ indicators and split-GFPs) allowed us to identify multiple partners of Trop-2 at the cell membrane.

Trop-2 and CD9 interaction revealed by tripartite split-GFP assay and confocal microscopy.

RNA interference and CRISPR/Cas9-based gene editing showed that Na+/K+ ATPase and CD9 are critical building blocks of a novel macromolecular membrane super-complex that drives cell growth and invasion.

CRISPR/Cas9 inactivation of CD9 (left) reduces the capability of Trop-2 to stimulate cell growth (middle) and invasion (right).

We then demonstrated that clustering of Trop-2, Na+/K+ ATPase and CD9 induces an intracellular Ca2+ rise followed by membrane translocation of PKCa, which in turn phosphorylates the Trop-2 cytoplasmic tail.

Clustering of Trop-2 at the plasma membrane induces intracellular Ca2+ rise (left)
followed by membrane recruitment of PKCα (right).

This novel feed-forward signaling axis induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the β-actin cytoskeleton through cofilin-1, and downstream activation of Akt and ERK.

Schematic representation of the Trop-2 signaling at the cell membrane
and the corresponding downstream effector cascade.

Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of colorectal cancer patients, supporting a pivotal relevance in colorectal cancer malignant progression and paving the way to design innovative strategies of targeted therapy.