Twist1 triggers PKD1 expression to promote metastatic dissemination in breast cancer
Authors: Georgess Dan , Sirka O. Katarina , Neumann Neil M. , Shamir Eliah R. , Ewald Andrew J. 
Published: November 2023

Metastasis is defined by a subset of cells disseminating from the epithelium, and eventually colonizing distant organs. The transcription factor Twist1 is overexpressed in metastatic breast cancers and its expression correlates with poor patient prognosis. Although Twist1 is known for promoting metastasis by controlling gene expression, its exact gene targets that are functionally involved in cell dissemination are still controversial.

To study Twist1 in the mammary epithelium, we used 3D organotypic culture of mammary epithelium fragments, called “organoids”, that we isolate from an inducible TetO‐Twist1 mouse. Induction of Twist1 expression in these organoids was sufficient to drive robust epithelial cell dissemination through extracellular matrix. RNAseq of Twist1‐induced and control organoids revealed 107 genes that are upregulated by Twist1 at genome‐wide significance. For 9 of these 107 genes, specific small molecule inhibitors were commercially available thus allowing for immediate dose‐response assay of dissemination. Inhibition of a single target – protein kinase D1 (PKD1) – with compound nb‐KB 142‐70 resulted in a significant decrease in dissemination. This result was surprising because PKD1 was previously thought to be expressed in normal breast epithelium and to be downregulated in breast cancer allowing tumor metastasis. However, our immunoblotting data showed that PKD1 is barely detectable in normal mammary organoids but is upregulated 7‐fold in Twist1‐induced organoids, while family member PKD2 was not detected in either. shRNA knockdown of PKD1 confirmed the outcome from its drug inhibition as it dramatically inhibited dissemination. Importantly, simultaneous targeting of PKD1 and of its direct upstream activator protein kinase C (PKC) with the compound Gö‐6976 completely blocked dissemination (IC50 11.5 nM). Gö‐6976's anti‐disseminative effect was significantly more potent than that of other kinase‐targeting drugs such as FDA‐approved Sorafenib; and, unlike Sorafenib, Gö‐6976 did not abolish normal branching morphogenesis of wildtype epithelium.

To validate these findings in human breast cancer, we organotypically cultured primary tumors surgically removed from patients, and we found that a single treatment with Gö‐6976 resulted in an overall 62% decrease of invasion (N= 6; 4 ER+/PR+ tumors and 2 ER‐/PR‐/Her2‐ tumors) and 86% decrease in dissemination (N=2). Independently from dissemination, Gö‐6976 also reduced colony formation potential of single cells isolated from a secondary/metastatic mammary tumor to the lymph node. Altogether, our data suggest a new working model where Twist1 can trigger PKD1 expression in primary mammary tumors, then PKD1 can drive distinct metastatic processes such as cell dissemination and proliferation.

Support or Funding Information

Dan Georgess is an American Association of Anatomists Scholar and he is funded by postdoctoral fellowships from the American Association of Anatomists and the Suzan G. Komen Foundation.