Identifying pathways regulating the oncogenic p53 family member ΔNp63 provides therapeutic avenues for squamous cell carcinoma

Abstract

Background ΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy. Methods We created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels. Results HaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells. Conclusions These data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

DNA Demethylation Switches Oncogenic ΔNp63 to Tumor Suppressive TAp63 in Squamous Cell Carcinoma

“…In conclusion, we have shown that inhibiting DNA methylation causes a switch in the relative levels of p63 isoforms, potentially converting ΔNp63-mediated tumor promotion and therapy resistance ( 7 – 9 , 12 , 13 ) towards TAp63-mediated tumor suppression ( 3 , 4 , 6 , 10 , 11 ). By identifying DNA hypermethylation at the TAP63 promoter in SCC, these data add this gene to the list of tumor suppressors that are epigenetically silenced in malignancy ( 40 – 42 ).…”

“…Plasmid DNAs were isolated using a Plasmid Maxi Kit (12162, Qiagen, Hilden, Germany) and used to produce viral particles in HEK293FT cells (ATCC). Lentiviruses were collected 48 and 96 h after transfection and transduced into HaCaT, FaDu and SCC-25 cells as described previously ( 12 ). Medium was replaced after 24 h, and selection in puromycin (1 µg/ml for HaCaT and FaDu and 0.05 µg/ml for SCC-25) started after a further 24 h. Medium containing puromycin was replaced every three days.…”

“…In particular, because TAp63 has p53-like properties, this approach is a viable option for replacing p53 tumor-suppressive activities in tumors with p53 mutation ( 10 , 11 ). Similarly, reducing the oncogenic activity of ΔNp63 causes tumor-suppressive activities for tumors that overexpress this protein ( 12 , 13 ).…”

“…Understanding the pathways involved in TAp63 and DNp63 regulation is therefore important to enable manipulation of their levels for cancer treatment. The aim of a p63-based therapeutic approach for tumors with high levels of DNp63 is to either decrease DNp63 or to increase TAp63, either of which can reduce tumor cell growth on their own (10)(11)(12)(13).…”

The promising therapeutic effects of metformin on metabolic reprogramming of cancer-associated fibroblasts in solid tumors

“…Metformin (1, 1-dimethyl biguanide) is a cost-effective and safe medication that helps diabetic patients to reduce their blood sugar levels. It has exhibited anti-tumorigenic effects in both experimental and clinical studies, suggesting a promising potential role in the treatment of multiple cancer treatments, such as ovarian cancer, lung adenocarcinoma, and breast cancer [ 87 – 90 ]. Metformin has been widely used by diabetic patients for over 40 years, but more recently a growing body of evidence suggests that metformin may also modulate the phenotype and the number of non-transformed cell types in the TME, such as CAFs, endothelial cells, and innate and adaptive immunity cells, including TAMs and T-lymphocytes, by multiple strategies [ 91 ].…”