Temporally resolved early BMP-driven transcriptional cascade during human amnion specification.

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that BMP signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hours after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification.

Spatially resolved cell polarity proteomics of a human epiblast model.

Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.

PRAS40 Phosphorylation Correlates with Insulin-Like Growth Factor-1 Receptor-Induced Resistance to Epidermal Growth Factor Receptor Inhibition in Head and Neck Cancer Cells.

EGFR inhibitors have shown poor efficacy in head and neck squamous cell carcinoma (HNSCC) with demonstrated involvement of the insulin-like growth factor-1 receptor (IGF1R) in resistance to EGFR inhibition. IGF1R activates the PI3K-Akt pathway, which phosphorylates proline-rich Akt substrate of 40 kDa (PRAS40) to cease mTOR inhibition resulting in increased mTOR signaling. Proliferation assays separated six HNSCC cell lines into two groups: sensitive to EGFR inhibition or resistant; all sensitive cell lines demonstrated reduced sensitivity to EGFR inhibition upon IGF1R activation. Reverse phase protein microarray analysis and immunoblot identified a correlation between increased PRAS40 phosphorylation and IGFR-mediated resistance to EGFR inhibition. In sensitive cell lines, PRAS40 phosphorylation decreased 44%-80% with EGFR inhibition and was restored to 98%-196% of control by IGF1R activation, while phosphorylation was unaffected in resistant cell lines. Possible involvement of mTOR in this resistance mechanism was demonstrated through a similar pattern of p70S6K phosphorylation. However, addition of temsirolimus, an mTORC1 inhibitor, was insufficient to overcome IGF1R-mediated resistance and suggested an alternative mechanism. Forkhead box O3a (FOXO3a), which has been reported to complex with PRAS40 in the cytoplasm, demonstrated a 6-fold increase in nuclear to cytoplasmic ratio upon EGFR inhibition that was eliminated with concurrent IGF1R activation. Transcription of FOXO3a-regulated TRAIL and PTEN-induced putative kinase-1 (PINK1) was increased with EGFR inhibition in sensitive cell lines; this effect was diminished with IGF1R stimulation. IMPLICATIONS: These data suggest PRAS40 may play an important role in IGF1R-based therapeutic resistance to EGFR inhibition, and this likely occurs via inhibition of FOXO3a-mediated proapoptotic gene transcription.

Antitumor effect of insulin-like growth factor-1 receptor inhibition in head and neck squamous cell carcinoma.

OBJECTIVES: The insulin-like growth factor-1 receptor (IGF1R) has been implicated in therapeutic resistance in head and neck squamous cell carcinoma (HNSCC), and small molecule tyrosine kinase inhibitors (TKIs) of IGF1R activity may have anticancer activity. Therefore, the relationship between survival and IGF1R expression was assessed for oral cavity (OC) cancer, and the antitumor effects of two IGF1R-TKIs, OSI-906 and BMS-754807, were evaluated in HNSCC cell lines in vitro. METHODS: Clinical outcome data and tissue microarray immunohistochemistry were used to generate IGF1R expression-specific survival curves. Immunoblot, alamarBlue proliferation assay, trypan blue exclusion viability test, clonogenic assay, flow cytometry, and reverse phase protein array (RPPA) were used to evaluate in vitro responses to IGF1R-TKIs. RESULTS: For patients with stage III/IV OCSCC, higher IGF1R expression was associated with poorer overall 5-year survival (P = 0.029). Both BMS-754807 and OSI-906 caused dose-dependent inhibition of IGF1R and Akt phosphorylation and inhibited proliferation; BMS-754807 was more potent than OSI-906. Both drugs reduced HNSCC cell viability; only OSI-906 was able to eliminate all viable cells at 10 µM. The two drugs similarly inhibited clonogenic cell survival. At 1 µM, only BMS-754807 caused a fourfold increase in the basal apoptotic rate. RPPA demonstrated broad effects of both drugs on canonical IGF1R signaling pathways and also inhibition of human epidermal growth factor receptor-3 (HER3), Src, paxillin, and ezrin phosphorylation. CONCLUSION: OSI-906 and BMS-754807 inhibit IGF1R activity in HNSCC cell lines with reduction in prosurvival and proliferative signaling and with concomitant antiproliferative and proapoptotic effects. Such antagonists may have utility as adjuvants to existing therapies for HNSCC. LEVEL OF EVIDENCE: NA Laryngoscope, 130:1470-1478, 2020.

Survivin in Insulin-Like Growth Factor-Induced Resistance to Lapatinib in Head and Neck Squamous Carcinoma Cells.

Epidermal growth factor receptor (EGFR) inhibitors have limited efficacy in head and neck squamous cell carcinoma (HNSCC) due to various resistance mechanisms, such as activation of the insulin-like growth factor-1 receptor (IGF1R), which initiates pro-survival signaling. Survivin, a member of the inhibitor of apoptosis proteins family, is expressed at relatively high levels in malignant tissues and plays a role in cell division. Expression of survivin in tumors has been shown to correlate with poor prognosis due to chemotherapy resistance and anti-apoptotic behavior. We previously demonstrated that activation of the IGF1R reduces sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) via reduced apoptosis suggesting a role of survivin in this process. This study evaluates the role of survivin in IGF1R-mediated lapatinib resistance. Using HNSCC cell lines FaDu and SCC25, survivin expression increased and lapatinib sensitivity decreased with IGF1R activation. Further, these effects were reversed by the survivin inhibitor YM-155. Conversely, survivin expression and lapatinib sensitivity were unchanged with IGF1R activation in UNC10 cells. YM-155 enhanced the inhibitory effect of lapatinib on UNC10 cells, regardless of activation of the IGF1R. These results demonstrate that enhanced survivin expression correlates with IGF1R-mediated lapatinib resistance in HNSCC cells and suggest that regulation of survivin expression may be a key mechanistic element in IGF1R-based therapeutic resistance. Combinatorial treatment with survivin antagonists and EGFR-TKIs warrants further investigation.

Activation of the insulin-like growth factor-1 receptor alters p27 regulation by the epidermal growth factor receptor in oral squamous carcinoma cells.

BACKGROUND: Although oral squamous cell carcinomas (OSCCs) commonly overexpress the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors (TKIs) exhibit poor efficacy clinically. Activation of the insulin-like growth factor-1 receptor (IGF1R) induces resistance of OSCC cells to EGFR-TKIs in vitro. This study seeks to evaluate the changes in cell cycle status in OSCC cells in response to gefitinib and IGF1R activation. METHODS: SCC-25 OSCC cells were used for in vitro analyses. RESULTS: Gefitinib caused a 50% reduction in S-phase population, and IGF1R activation caused a 2.8-fold increase; combined treatment yielded a baseline S-phase population. Gefitinib treatment increased the cyclin-dependent kinase inhibitor p27, and this was not abrogated by IGF1R activation. pT157-p27 was noted by immunoblot to be decreased on gefitinib treatment, but this was reversed with IGF1R activation. T157 phosphorylation contributes to cytoplasmic localization of p27 where it can promote cell proliferation and cell motility. Using both subcellular fractionation and immunofluorescence microscopy techniques, IGF1R stimulation was noted to increase the relative cytoplasmic localization of p27; this persisted when combined with gefitinib. CONCLUSIONS: IGF1R activation partially reverses the cell cycle arrest caused by gefitinib in OSCC cells. While IGF1R stimulation does not eliminate the gefitinib-induced increase in total p27, its phosphorylation state and subcellular localization are altered. This may contribute to the ability of the IGF1R to rescue OSCC cells from EGFR-TKI treatment and may have important implications for the use of p27 as a biomarker of cell cycle arrest and response to therapy.

Activation of the insulin-like growth factor-1 receptor induces resistance to epidermal growth factor receptor antagonism in head and neck squamous carcinoma cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have poor efficacy in head and neck squamous carcinoma cells (HNSCC). Because the IGF-1 receptor (IGF1R) generates potent prosurvival signals and has been implicated in therapeutic resistance, its ability to induce resistance to EGFR-TKIs was studied in vitro. Five HNSCC cell lines showed reduced sensitivity to the EGFR-TKI gefitinib when the IGF1R was activated. In SCC-25 and Cal27 cells, gefitinib inhibited basal and EGF-stimulated EGFR, extracellular signal-regulated kinase (Erk), and Akt phosphorylation and reduced cell number. This correlated with initiation of apoptosis based on a 4-fold increase in PARP cleavage and a 2.5-fold increase in Annexin V positivity. The apoptotic response and reduction in cell number were blocked by IGF1R activation, which resulted in phosphorylation of both Erk and Akt. In both the cell lines, IGF1R-induced Erk, but not Akt, activation was eliminated by gefitinib. IGF1R-induced gefitinib resistance was unaffected by MAP/Erk kinase inhibition with U0126 but was partially impaired by inhibition of phosphoinositide-3-kinase with LY294002. The IGF1R-TKI PQ401 inhibited growth of SCC-25 and Cal27 cells alone and also acted synergistically with gefitinib. Thus, the IGF1R can make HNSCC cells resistant to EGFR-TKI treatment via a prosurvival mechanism. Of the 8 HNSCC tumor samples studied, all samples expressed the IGF1R and 5 showed detectable IGF1R phosphorylation, suggesting that this receptor may be relevant in vivo, and thus, combined EGFR/IGF1R inhibition may be necessary in some patients for effective targeted molecular therapy.