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Radiotherapy plays an essential role in the treatment of head and neck squamous cell carcinoma (HNSCC), yet radioresistance remains a major barrier to therapeutic efficacy. A better understanding of the predominant pathways determining radiotherapy response could help develop mechanism-informed therapies to improve cancer management. Here we report that radioresistant HNSCC cells exhibit increased tumor aggressiveness. Using unbiased proteome profiler antibody arrays, we identify that upregulation of c-Met phosphorylation is one of the critical mechanisms for radioresistance in HNSCC cells. We further uncover that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but is suppressed by its genetic knockdown and pharmacologic inactivation. Mechanistically, the resulting upregulation of c-Met promotes elevated expression of plexin domain containing 2 (PLXDC2) through activating ERK1/2-ELK1 signaling, which in turn modulates cancer cell plasticity by epithelial–mesenchymal transition (EMT) induction and enrichment of the cancer stem cell (CSC) subpopulation, leading to resistance of HNSCC cells to radiotherapy. Depletion of PLXDC2 overcomes c-Met–mediated radioresistance through reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, the addition of SU11274, a selective and potent c-Met inhibitor, to radiation induces tumor shrinkage and limits tumor metastasis to lymph nodes in an orthotopic mouse model. Collectively, these significant findings not only demonstrate a novel mechanism underpinning radioresistance-associated aggressiveness but also provide a possible therapeutic strategy to target radioresistance in patients with HNSCC. Significance: This work provides novel insights into c-Met-PLXDC2 signaling in radioresistance-associated aggressiveness and suggests a new mechanism-informed therapeutic strategy to overcome failure of radiotherapy in patients with HNSCC.

Coronary microvascular dysfunction contributes to the development of left ventricular (LV) diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF), a prevalent symptom that has limited targeted treatment options. Selective pharmacological inhibitors of phosphodiesterase 9A (PDE9A), such as PF04447943, have emerged as a potential therapeutic approach for the treatment of cardiometabolic diseases, in part, owing to their antioxidant effects. We hypothesized that PDE9A inhibition has beneficial effects on the coronary microvasculature, thereby improving LV diastolic function in HFpEF. In order to study this, the Obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat, a model of human HFpEF, was employed in which it was found that progressively developing coronary microvascular rarefaction, as quantified by an unbiased, three-dimensional reconstruction of the coronary microvascular network (Vesselucida360), as well as vasodilator coronary arteriole dysfunction are associated with the development of LV diastolic dysfunction, which were improved by the treatment with the selective PDE9A inhibitor, PF04447943 (3 mg/kg/day, oral gavage for 2 weeks). These coronary microvascular changes were accompanied by an increase in cardiac 3-nitrotyrosine, a marker of nitrosative stress, which was also mitigated by PF04447943 treatment. Furthermore, mass spectrometry-based proteomics and pathway analyses identified peroxiredoxin-5 (PRDX5) as one of the predominantly downregulated antioxidants in the heart of the obese ZSF1 rats, which was augmented after PDE9A inhibition. From this data, it was concluded that PDE9A inhibition, potentially through augmenting the PRDX-5 antioxidant mechanism, improved coronary vascular rarefaction and vasodilator function, thereby restoring LV diastolic function in obese ZSF1 rats. T32HL155011 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The disintegrin and metalloproteinase 17 (ADAM17) exhibits α-secretase activity, whereby it can prevent the production of neurotoxic amyloid precursor protein-α (APP). ADAM17 is abundantly expressed in vascular endothelial cells and may act to regulate vascular homeostatic responses, including vascular growth and vasomotor function. We hypothesized that cerebrovascular ADAM17 plays a role in the pathogenesis of Alzheimer’s disease (AD). Here we found that 8-10 months old APP/PS1 mice (expressing transgenes for APP Swedish mutation and PSEN1 with L166P mutation) with significant cognitive deficits display a markedly reduced expression of ADAM17 in cerebral microvessels. Systemic delivery and AAV-mediated re-expression of ADAM17 in APP/PS1 mice improved cognitive functioning, without affecting β-amyloid plaque density. Moreover, we found that the endothelium-dependent vasodilator function of isolated and pressurized cerebral arteries in APP/PS1 mice was impaired, which was restored to normal levels by the ADAM17 re-expression. The cerebral microvascular network density in the APP/PS1 mice before or after ADAM17 re-expression was not affected. In addition, proteomic analysis identified several differentially expressed molecules involved in AD neurodegeneration and repair that were reversed by ADAM17 re-expression. Thus, we propose that a reduced ADAM17 expression in cerebral microvessels impairs vasodilator function, whereby it contributes to the development of cognitive dysfunction in APP/PS1 mice, and that ADAM17 can potentially be targeted for therapeutic intervention in AD. T32HL155011; 20PRE35211126 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Accumulating epidemiological evidence shows that the incidence of multiple types of cancer, such as melanoma and GI cancers, is significantly higher in patients with heart failure with preserved ejection fraction (HFpEF). However, the mechanistic relationships between HFpEF and cancer development are poorly understood. Impaired angiogenesis, upregulation of endothelial adhesion molecules and chronic low-grade inflammation are common in HFpEF. Here we examined the role of endothelial cell-selective adhesion molecule (ESAM) in the progression of malignant melanoma in a HFpEF mouse model. Orthotopic melanoma xenografts (B16F10-Luc2) were used and implanted subcutaneously in mice underwent uninephrectomy and aldosterone infusion (UNX-Aldo with 1% high salt drinking water for 4 weeks) and tumor growth was monitored with bioluminescent imaging. We found that ESAM expression and serum levels were elevated in the UNX-Aldo mice, which was accompanied by enhanced melanoma growth and metastasis rate. ESAM promoted tube formation and increased phospho-Akt, phospho-PKCα and phospho-ERK1/2 in cultured endothelial cells. Flow cytometry analysis showed elevated number of tumor-associated neutrophils (CD11b+Ly6G+Ly6C-) and myeloid-derived suppressor cells (MDSCs, CD11b+F4/80-Gr-1+) in the UNX-Aldo mice. UNX-Aldo mice with genetic deletion of ESAM (ESAM KO) showed a reduced tumor growth and reduced number of tumor-associated neutrophils and MDSCs. Collectively, our results indicate that in HFpEF, a proinflammatory ESAM activation may act to promote tumor angiogenesis, neutrophil and MDSCs tumor infiltration, thereby mitigating a proper immune response and facilitate tumor growth. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of PLXDC2 knockdown on colony formation of radioresistant CAL27 and HN6 cells determined on Day 12 following 4 Gy IR exposure

Radiotherapy plays an essential role in the treatment of head and neck squamous cell carcinoma (HNSCC), yet radioresistance remains a major barrier to therapeutic efficacy. A better understanding of the predominant pathways determining radiotherapy response could help develop mechanism-informed therapies to improve cancer management. Here we report that radioresistant HNSCC cells exhibit increased tumor aggressiveness. Using unbiased proteome profiler antibody arrays, we identify that upregulation of c-Met phosphorylation is one of the critical mechanisms for radioresistance in HNSCC cells. We further uncover that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but is suppressed by its genetic knockdown and pharmacologic inactivation. Mechanistically, the resulting upregulation of c-Met promotes elevated expression of plexin domain containing 2 (PLXDC2) through activating ERK1/2-ELK1 signaling, which in turn modulates cancer cell plasticity by epithelial–mesenchymal transition (EMT) induction and enrichment of the cancer stem cell (CSC) subpopulation, leading to resistance of HNSCC cells to radiotherapy. Depletion of PLXDC2 overcomes c-Met–mediated radioresistance through reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, the addition of SU11274, a selective and potent c-Met inhibitor, to radiation induces tumor shrinkage and limits tumor metastasis to lymph nodes in an orthotopic mouse model. Collectively, these significant findings not only demonstrate a novel mechanism underpinning radioresistance-associated aggressiveness but also provide a possible therapeutic strategy to target radioresistance in patients with HNSCC.Significance:This work provides novel insights into c-Met-PLXDC2 signaling in radioresistance-associated aggressiveness and suggests a new mechanism-informed therapeutic strategy to overcome failure of radiotherapy in patients with HNSCC.

The primer sequences for qRT-PCR assays.

Physiological and pathological vascular remodeling is uniquely driven by mechanical forces from blood flow in which wall shear stress (WSS) mechanosensing by the vascular endothelium plays a pivotal role. This study aimed to determine the novel role for a disintegrin and metalloproteinase 17 (ADAM17) in impaired WSS mechanosensing, which was hypothesized to contribute to aging-associated abnormal vascular remodeling. Without changes in arterial blood pressure and blood flow rate, skeletal muscle resistance arteries of aged mice (30-month-old vs. 12-week-old) exhibited impaired WSS mechanosensing and displayed inward hypertrophic arterial remodeling. These vascular changes were recapitulated by in vivo confined, AAV9-mediated overexpression of ADAM17 in the resistance arteries of young mice. An aging-related increase in ADAM17 expression reduced the endothelial junction level of its cleavage substrate, junctional adhesion molecule-A/F11 receptor (JAM-A/F11R). In cultured endothelial cells subjected to steady WSS ADAM17 activation or JAM-A/F11R knockdown inhibited WSS mechanosensing. The ADAM17-activation induced, impaired WSS mechanosensing was normalized by overexpression of ADAM17 cleavage resistant, mutated JAM-A(V232Y) both in cultured endothelial cells and in resistance arteries of aged mice, in vivo. These data demonstrate a novel role for ADAM17 in JAM-A/F11R cleavage-mediated impaired endothelial WSS mechanosensing and subsequently developed abnormal arterial remodeling in aging. ADAM17 could prove to be a key regulator of WSS mechanosensing, whereby it can also play a role in pathological vascular remodeling in diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-021-00476-1.

Head and neck squamous cell carcinoma (HNSCC) remains a deadly disease despite concerted efforts to improve its diagnosis and treatment in recent decades. Metastasis of advanced HNSCC nearly always occurs first in neck lymph nodes before the development of distant metastasis. However, the development of preclinical animal models and therapeutic treatments for metastatic HNSCC is lagged from bench to clinic. In this protocol, we exemplify an orthotopic tongue tumor model that can recapitulate the cervical lymphatic metastases of HNSCC and the application to study the effect of novel saracatinib-loaded nanoparticles (Nano-Sar). By taking advantage of bioluminescence imaging (BLI), the present protocol reveals the strong anti-metastatic efficacy of Nano-Sar in the experimental setup. Collectively, the protocol with a novel metastatic mouse model shows great potential to evaluate treatments on metastatic diseases with the aid of bioluminescent technology.

The development of radioresistance in head and neck squamous cell carcinoma (HNSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in the past decades. One of the clinical challenges is that radioresistance often promotes tumor aggressiveness. However, the underlying mechanisms and molecular determinants are largely unknown. We report here that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but can be suppressed by its genetic knockdown and pharmacological inactivation. Through unbiased RNAseq data, we further uncovered that the resulting upregulation of c-Met signaling increases the expression of PLXDC2. a critical gene associated with the tumor microenvironment. Mechanistically, PLXDC2 is upregulated in radioresistant HNSCC cells through c-Met mediated activation of ERK1/2-ELK1 signaling cascade. Most importantly, PLXDC2 modulates cancer cell plasticity by inducing epithelial-mesenchymal transition (EMT) and the emergence of cancer stem cell (CSC) subpopulation. Additionally, depletion of PLXDC2 overcomes c-Met-mediated radioresistance by reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, a combination of the c-Met selective inhibitor SU11274 with radiation remarkably induces tumor shrinkage and constrains tumor metastasis to lymph nodes in vivo. Our study is novel in being the first to explore the role and mechanism of the c-Met-PLXDC2 axis in radioresistance-associated HNSCC aggressiveness and the first to our knowledge to evaluate how to take advantage of blocking this signaling to overcome radioresistance in preclinical mouse models of HNSCC. Citation Format: Fanghui Chen, Liwei Lang, Chloe Shay, Georgia Chen, Nabil Saba, Yong Teng. Met confers radioresistance-associated aggressiveness through enhancing PLXDC2-mediated cancer stem cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5793.

Numerous studies have clarified the effectiveness of paeoniflorin in the treatment of cholestasis. However, the therapeutic efficacy and mechanisms of action of paeoniflorin in intrahepatic cholestasis of pregnancy (ICP) were still unknown. This study aimed to investigate the molecular biological mechanisms of paeoniflorin against ICP by combining network pharmacology and metabolomics. The effects of paeoniflorin were investigated in the ICP rat model induced by 17α-ethinylestradiol, showing improvements in the liver indices, liver histopathological changes, bile flow rate, and serum levels of TBA and ALP. The underlying mechanisms and metabolic pathways of paeoniflorin were revealed by network pharmacology and untargeted metabolomics, showing that paeoniflorin exerted its curative effect against ICP-induced ferroptosis through PI3K/AKT and MAPK signalling pathways. In conclusion, paeoniflorin protected against ICP-induced liver injury through MAPK signaling pathways.

The identification and development of reagents that can limit preferential metabolic effectors in tumors as well as trigger unfavorable tumoral metabolic cues is of tremendous importance for suppressing malignancy. However, insufficient blocking of critical metabolic dependencies of cancer allows the development of metabolic bypasses, which significantly dampens therapeutic benefits of selective agents that target a single metabolic enzyme or pathway. We report here that head and neck squamous cell carcinoma (HNSCC) cells display strong addiction to glutamine. Devimistat, a novel lipoate analog, redirects cellular activity towards tumor-promoting glutaminolysis, leading to low anticancer efficacy in HNSCC cells. Mechanistically, devimistatinhibits the tricarboxylic acid cycle by blocking the enzyme activities of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which upregulates GLS1 and eventually promotes the compensatory role of glutaminolysis in cancer cell survival. Most importantly, the addition of a GLS1 inhibitor CB-839 to devimistat treatment abrogates the metabolic dependency of HNSCC cells on glutamine, achieving a synergistic anticancer effect in glutamine-addicted HNSCC. These novel and significant findings could lay a scientific foundation for developing more effective treatments targeting the metabolic requirements of HNSCC. Citation Format: Liwei Lang, Fang Wang, Chloe Shay, Yonggang Ke, Nabil Saba, Yong Teng. Inhibition of glutaminolysis overcomes metabolic adaptation to devimistat treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3026.

Targeting mitochondria as a cancer therapeutic strategy has attracted more and more attention in the recent years. ATAD3A is identified as a nuclear-encoded mitochondrial enzyme that regulates mitochondrial dynamics, cholesterol metabolism, and signal transduction. However, its impact and underlying regulatory mechanisms in cancers remain to be determined. We report here that ATAD3A is highly expressed in head and neck squamous cell carcinoma (HNSCC), where it plays a critical role in favoring tumor progression. Loss of ATAD3A expression dramatically incudes tumor regression in orthotopic tongue tumor-bearing mice, whereas gain of ATAD3A expression produces the opposite effects. Mechanistically, ATAD3A binds to ERK1/2 at HNSCC cell mitochondria in the presence of VDAC1, and this interaction is essential for the activation of mitochondrial ERK1/2 signaling. Most importantly, the ATAD3A-ERK1/2 signaling axis enhances HNSCC development in a Ras-independent manner and, thus, tumor suppression is more effectively achieved when ATAD3A and Ras signaling pathways are co-inhibited. These novel findings highlight the importance of activation of ATAD3A-ERK1/2 axis in cancer progression and suggest that combined targeting of ATAD3A and Ras signaling could potentiate anticancer activity for HNSCC treatment. Citation Format: Liwei Lang, Reid Loveless, Zhaohui Qin, Peng Qiu, Chloe Shay, Yong Teng. ATAD3A enhances head and neck cancer malignancy via mitochondrial ERK1/2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 88.

One of the significant clinical challenges in head and neck cancer (HNC) treatment is a high rate of recurrence and metastasis after radiotherapy. Once tumor metastasis occurs following radiotherapy, the prognosis of HNC patients is extremely poor. However, the failure of radiotherapy for cancers is closely related to various factors, and the underlying mechanism remains unclear. We report here that c-Met is the most activated receptor tyrosine kinases in HNC cells once they develop radioresistance. Inactivation of c-Met dramatically attenuates the increased metastatic potential gained in HNC radioresistant cells, leading to tumor regression. Cancer stem cells (CSC) have been confirmed to be in a quiescent state in most established tumors. Mechanistically, the quiescent CSC population can be “awakened” by hyperactive c-Met signaling in radiation, which in turn initiates proliferation and differentiation to potentiate a series of malignant phenotypes. Most importantly, knockdown c-Met by shRNAs or inactivating it by c-Met inhibitor SU11274 reduces the population of “awakened” CSCs and suppresses metastasis, leading to increased sensitivity of HNC radioresistant cells to radiation. These novel findings highlight the importance of additional c-Met blockade in radiotherapy and suggest that combined targeting of c-Met and radiotherapy could be a promising strategy to improve the care for patients with HNC. Citation Format: Juan Dou, Liwei Lang, Georgia Chen, Nabil F. Saba, Yong Teng. Blockade of c-met signaling to circumvent radioresistance-associated metastasis in head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B038.