AP-1 and TGFß cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.


Palaeontology and archaeology
Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Validation Sample sizes were chosen for at least n = 3 biological replicates. Exact sample sizes are noted in the Methods section. No sample size calculation was performed. Enough replicates were provided to allow for p-value calculations.
No data were excluded from the analyses.
Experiments were repeated at least 3 times independently. When possible, alternative methods (different inhibitors, siRNA oligo sequences, etc) were used to confirm specificity of effects. All attempts at replication were successful.
Samples were randomly distributed into treatment or control groups and experiments were carried out in parallel.
Investigator was not blinded to group allocation because the same investigator conducted data collection as well as analysis.

Human research participants
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Recruitment
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ChIP-seq Data deposition
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No commonly misidentified cell lines were used in the study.
No wild animals were used in the study.
No field collected samples were used in the study.
The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University.
Patient population included patients with diagnosed basal cell carcinoma with no prior treatment. Samples were deidentified and there was no selection for age, gender, or other characteristics.