Epidermal Loss of Gag Confers a Migratory and Differentiation Defect in Keratinocytes

G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gaq and Ga-11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Ga-11 and Gaq from endothelial cells impairs the Rho -mediated formation of focal adherens junctions, suggesting that Gai vg signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Ga-11 and Gaq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gaq-null keratinocytes under combined Ga-11 deficiency. In fact, cells lacking Gaivg demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gaivg signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gai vg signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions

Characterization of the Immune Response to PD-1 Blockade during Chemoradiotherapy for Head and Neck Squamous Cell Carcinoma

Background: Chemoradiotherapy is a standard treatment for HNSCC. Blockade of the PD-1/L1-2 interaction may represent a target to overcome immune escape during this treatment. Methods: Utilizing a HNSCC mEERL C57BL/6 mouse model, we evaluated a PD-1 blockade alone or in combination with cisplatin-based chemoradiotherapy. Next, we evaluated peripheral blood mononuclear cells (PBMCs) with relative PD-1, TIM-3, and LAG-3 expression, and myeloid-derived suppressor-like (MDSC-like) populations from a clinical trial evaluating PD-1 blockade with chemoradiotherapy in HNSCC. Finally, we analyzed the effect of therapy on human T-cell clonality through T-cell Receptor (TCR) sequencing. Results: Anti-PD-1 monotherapy induced no response in the mEERL model; however, combination with chemoradiotherapy improved tumor clearance and survival. PBMCs from patients treated with this combination therapy demonstrate a decline in circulating T-cell populations with knockdown of PD-1 expressing CD3+CD4+ and CD3+CD8+ T cells during treatment. However, TIM-3, LAG-3 expressing T-cell and MDSC-like populations concordantly rose. During treatment, the TCR repertoire demonstrates overall clonal expansion, with both unique and previously reported T-cell clones. Conclusions: Our murine HNSCC model demonstrates efficacy of PD-1 blockade during chemoradiotherapy. However, while PD-1-expressing T cells decreased with this therapy, human PBMC findings also identified an increase in populations contributing to immune exhaustion. These findings further characterize PD-1 blockade during chemoradiotherapy for HNSCC and highlight potential competing mechanisms of immune evasion

Characterization of the Immune Response to PD-1 Blockade during Chemoradiotherapy for Head and Neck Squamous Cell Carcinoma

Background: Chemoradiotherapy is a standard treatment for HNSCC. Blockade of the PD-1/L1-2 interaction may represent a target to overcome immune escape during this treatment. Methods: Utilizing a HNSCC mEERL C57BL/6 mouse model, we evaluated a PD-1 blockade alone or in combination with cisplatin-based chemoradiotherapy. Next, we evaluated peripheral blood mononuclear cells (PBMCs) with relative PD-1, TIM-3, and LAG-3 expression, and myeloid-derived suppressor-like (MDSC-like) populations from a clinical trial evaluating PD-1 blockade with chemoradiotherapy in HNSCC. Finally, we analyzed the effect of therapy on human T-cell clonality through T-cell Receptor (TCR) sequencing. Results: Anti-PD-1 monotherapy induced no response in the mEERL model; however, combination with chemoradiotherapy improved tumor clearance and survival. PBMCs from patients treated with this combination therapy demonstrate a decline in circulating T-cell populations with knockdown of PD-1 expressing CD3+CD4+ and CD3+CD8+ T cells during treatment. However, TIM-3, LAG-3 expressing T-cell and MDSC-like populations concordantly rose. During treatment, the TCR repertoire demonstrates overall clonal expansion, with both unique and previously reported T-cell clones. Conclusions: Our murine HNSCC model demonstrates efficacy of PD-1 blockade during chemoradiotherapy. However, while PD-1-expressing T cells decreased with this therapy, human PBMC findings also identified an increase in populations contributing to immune exhaustion. These findings further characterize PD-1 blockade during chemoradiotherapy for HNSCC and highlight potential competing mechanisms of immune evasion

Epidermal loss of Gαq confers a migratory and differentiation defect in keratinocytes.

G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gαq and Gα11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Gα11 and Gαq from endothelial cells impairs the Rho-mediated formation of focal adherens junctions, suggesting that Gα11/q signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Gα11 and Gαq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gαq-null keratinocytes under combined Gα11 deficiency. In fact, cells lacking Gα11/q demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gα11/q signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gα11/q signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions

Gα₁₁KO/Gα_q-eKO mice have delayed wound healing.

<p>Gα₁₁KO/Gα_q-WT control and Gα₁₁KO/Gα_q-eKO mice received 15 mm incisional wound and closure was monitored over 18 days. A) Gross examination of the wounds in Gα₁₁KO/Gα_q-WT control (N = 15) and Gα₁₁KO/Gα_q-eKO (N = 10) mice. B) Initial wound size. Statistical significance determined by Student’s t-test. C) Kaplan-Meier survival curve of wound closure. Statistical significance was determined by log-rank test. D) Wound closure over time. Statistical significance was determined by two-way ANOVA. E) Wound half-life for Gα₁₁KO/Gα_q-WT control and Gα₁₁KO/Gα_q-eKO animals, as determined from the rate of wound closure over time. Statistical significance was determined by Student’s t-test, ***p<0.001.</p

Gα_11/q-null keratinocytes have normal proliferative and stem-like phenotypes but demonstrate a significant migratory defect.

<p>Keratinocytes from Gα₁₁KO/Gα_q-WT control and Gα₁₁KO/Gα_q-eKO animals were assayed for proliferation (A), colony formation (B) and scratch wound closure (C). To confirm the migratory defect, keratinocyte and fibroblast wound closure from Gα₁₁KO/Gα_q-WT control and Gα₁₁KO/Gα_q-eKO were quantified. Statistical significance was determined using Student’s t-test, *p<0.05, **p<0.01, ***p<0.001.</p

Loss of epidermal Gα_q correlates with defects in reepithelialization of cutaneous wounds.

<p>Top panel: the epithelial tongue as evidenced by H&E staining (yellow dashed line) is diagrammed and the length quantified. Representative stains and quantification of Ki67 (second panel), K10 (third panel), and K13 (bottom panel) are shown. All quantifications are based on an N = 4 mice per group and proliferation is shown relative to normal adjacent epithelium. Slope was determined by linear regression and statistical significance was determined by Student’s t-test, p<0.05, **p<0.01, ***p<0.001.</p

Balance between MKK6 and MKK3 mediates p38 MAPK associated resistance to cisplatin in NSCLC.

The p38 MAPK signaling pathway has been proposed as a critical mediator of the therapeutic effect of several antitumor agents, including cisplatin. Here, we found that sensitivity to cisplatin, in a system of 7 non-small cell lung carcinoma derived cell lines, correlated with high levels of MKK6 and marked activation of p38 MAPK. However, knockdown of MKK6 modified neither the response to cisplatin nor the activation of p38 MAPK. Deeper studies showed that resistant cell lines also displayed higher basal levels of MKK3. Interestingly, MKK3 knockdown significantly decreased p38 phosphorylation upon cisplatin exposure and consequently reduced the response to the drug. Indeed, cisplatin poorly activated MKK3 in resistant cells, while in sensitive cell lines MKK3 showed the opposite pattern in response to the drug. Our data also demonstrate that the low levels of MKK6 expressed in resistant cell lines are the consequence of high basal activity of p38 MAPK mediated by the elevated levels of MKK3. This finding supports the existence of a regulatory mechanism between both MAPK kinases through their MAPK. Furthermore, our results were also mirrored in head and neck carcinoma derived cell lines, suggesting our observations boast a potential universal characteristic in cancer resistance of cisplatin. Altogether, our work provides evidence that MKK3 is the major determinant of p38 MAPK activation in response to cisplatin and, hence, the resistance associated with this MAPK. Therefore, these data suggest that the balance between both MKK3 and MKK6 could be a novel mechanism which explains the cellular response to cisplatin