Basal cell carcinoma (BCC) is the most common malignancy in humans. We present a man with a recurrent BCC of the scalp that presented as an intracranial tumor 18 years after original excision.

Basal cell carcinoma (BCC) is driven by overactivation of the Hedgehog signaling pathway whose current FDA-approved chemical inhibitors can efficiently suppress protein receptor Smoothened (SMOi) but have presented serious resistance problems in clinical settings. In SMOi-resistant BCCs, we documented an important correlation between the increase in mRNA level of the constitutively active isoform of atypical protein kinase C (aPKC-K) and the surge in mutational load of ciliary proteins. We demonstrated, for the first time, that aPKC protein expression was upregulated upon cilia loss and that chemical inhibition of aPKC increased both percent ciliation and cilia shaft length, both in cells and BCC mice. We also found that the Hedgehog pathway activity (a) was significantly diminished when ciliogenesis was disrupted by transient knockdown of important ciliary genes and (b) was adequately rescued when aPKC-K was simultaneously overexpressed. At the same time, cells that overexpressed aPKC-K displayed resistance to Vismodegib, a widely prescribed SMOi. We also recorded higher aPKC protein expression and lower ciliogenesis in human superficial BCC subtype in comparison to nodular BCC subtype, suggesting a connection between the interfollicular epidermal origin of superficial BCCs and the SMOi-resistance tendency, presumably via aPKC overactivation coupled with cilia loss. In a second project, we detailed the functional effects of phosphorylation by protein kinase A (PKA) at individual and combinatorial PKA-specific phosphosites on GLI1. Artificial loss of PKA-phosphorylation promoted growth in murine BCC cells and allografts; whereas overexpression of clinical mutations found in resistant BCCs near the PKA-sites on GLI1 altered GLI1 activity, localization, and stability. We also described, for the first time, a truncated protein product of GLI1 when PKA-phosphorylation was mimicked. These findings challenged the currently accepted notion that GLI1 could only be completely degraded by the βTrCP/proteasome complex. Instead, these results suggest that PKA may negate GLI1 activity in a graded fashion via specific phosphorylation at the three C-terminal PKA-phosphosites. Therefore, we propose two new mechanisms of SMOi-resistance in BCCs: (1) selecting for ciliary mutations that abolish the primary cilia-dependent canonical HH pathway while overactivating aPKC to non-canonically promote downstream GLI transcription factor, and (2) selecting for GLI1 mutations that disrupt the inactivating phosphorylation by PKA to maintain canonical HH signaling.

Basal cell carcinoma is the most prevalent cancer and it has been well-documented to be driven predominantly via overactivation of the Hedgehog signaling pathway. Although vismodegib, a SMO inhibitor, has been proven to be highly effective in treating BCCs, advanced forms of BCCs often possess inherent resistance while many that initially respond to drug treatment develop drug resistance over time. This highlights the importance of identifying targets that are either downstream of SMO or alternative drivers of BCCs in order to bypass SMO-inhibitor resistance. Here, we use a combination of RNA-sequencing and immunofluorescence staining to identify and validate the overexpression of other potential pathways that are upregulated in BCCs. This analysis highlighted the expression of mTOR, PI3K, and SRC. We found that upon pharmacological inhibition of mTOR and PI3K in our Ptch1fl/fl; Gli1-CreERT2 mouse BCC tumor model that although BCC growth is significantly inhibited, HH signaling is not. This implies that both the mTOR and PI3K pathway may work either downstream of or in parallel with HH signaling. Furthermore, our data suggests that mTOR is affecting BCC growth via aPKC independent of HH signaling whereas PI3K is likely driving BCC growth via aPKC- and AKT-driven p21 degradation. However, when SRC was pharmacologically inhibited, it reduced both BCC growth and HH signaling. We were able to demonstrate that SRC promotes BCC growth via aPKC-dependent phosphorylation and activation of GLI1. Together, these findings identify alternative means of being able to target and treat BCCs and, potentially, resistant BCCs as well.