Search

Oral squamous cell carcinoma (OSCC) has 50% 5-year survival rate, highlighting our limited understanding of the molecular events that contribute to disease progression. Microarray analyses of primary oral tumors have identified urinary-type plasminogen activator (uPA) and its receptor (uPAR) as key genes associated with human OSCC progression. The uPAR functions as both a proteinase receptor and an integrin ligand, modifying proteolysis, migration, integrin signaling, and cellular transcription. In the current study, uPAR expression levels were modified in OSCC cells followed by analysis of tumor growth in an in vivo orthotopic xenograft model and by transcriptional profiling. Overexpression of uPAR resulted in more infiltrative and less differentiated tumors, with ill-defined borders, cytologic atypia, and enhanced vascularity. Analysis of serial sections of both murine experimental tumors and microarrayed human OSCC showed a statistically significant association between uPAR and α3 integrin colocalization in areas exhibiting extracellular signal-regulated kinase phosphorylation, suggesting that uPAR/α3 integrin interaction potentiates extracellular signal-regulated kinase signaling in vivo. This is supported by cDNA microarray analysis, which showed differential expression of 148 genes (113 upregulated and 35 downregulated). Validation of gene expression changes in human OSCC using immunohistochemistry and quantitative real-time PCR showed increased growth factors, proteinases/inhibitors, and matrix components in uPAR-overexpressing tumors. Together, these results support a model wherein increased uPAR expression promotes α3β1 integrin association, resulting in increased mitogen-activated protein kinase signaling and transcriptional activation, leading to the formation of more aggressive tongue tumors. This combined approach has efficacy to identify additional biomarkers and/or prognostic indicators associated with aggressive human OSCC. Mol Cancer Res; 8(2); 145–58

Supplementary Data from Urinary-Type Plasminogen Activator Receptor/α3β1 Integrin Signaling, Altered Gene Expression, and Oral Tumor Progression

Therapeutic outcomes achieved in head and neck squamous cell carcinoma (HNSCC) patients by concurrent cisplatin-based chemoradiotherapy initially reflect both tumor regression and tumor stasis. However, local and distant metastasis and disease relapse are common in HNSCC patients. In the current work, we demonstrate that cisplatin treatment induces senescence in both p53 wild-type HN30 and p53 mutant HN12 head and neck cancer models. We also show that tumor cells can escape from senescence both in vitro and in vivo. We further establish the effectiveness of the senolytic, ABT-263 (Navitoclax), in elimination of senescent tumor cells after cisplatin treatment. Navitoclax increased apoptosis by 3.3-fold (P ≤ 0.05) at day 7 compared with monotherapy by cisplatin. Additionally, we show that ABT-263 interferes with the interaction between B-cell lymphoma-x large (BCL-X(L)) and BAX, anti- and pro-apoptotic proteins, respectively, followed by BAX activation, suggesting that ABT-263-induced apoptotic cell death is mediated through BAX. Our in vivo studies also confirm senescence induction in tumor cells by cisplatin, and the promotion of apoptosis coupled with a significant delay of tumor growth after sequential treatment with ABT-263. Sequential treatment with cisplatin followed by ABT-263 extended the humane endpoint to ∼130 days compared with cisplatin alone, where mice survived ∼75 days. These results support the premise that senolytic agents could be used to eliminate residual senescent tumor cells after chemotherapy and thereby potentially delay disease recurrence in head and neck cancer patients. SIGNIFICANCE STATEMENT: Disease recurrence is the most common cause of death in head and neck cancer patients. B-cell lymphoma-x large inhibitors such as ABT-263 (Navitoclax) have the capacity to be used in combination with cisplatin in head and neck cancer patients to eliminate senescent cells and possibly prevent disease relapse.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and disproportionately affects BRCA1 mutation carriers and young black women. Black/African American (AA) patients with TNBC have the highest mortality rate and the shortest survival of any racial/ethnic group in the United States. TNBC challenges our ability to personalize effective therapy. Neoadjuvant chemotherapy (NACT) is the standard-of-care (SOC) to treat T2 or LN+ TNBC. The addition of immune checkpoint blockade (ICB) has revolutionized treatment for these high-risk patients. Pembrolizumab plus chemotherapy is the new SOC for neoadjuvant systemic therapy (NST) in TNBC. Pathologic complete response (pCR) is a reliable indicator that correlates with improved long-term survival. However, clinical uncertainties remain in those who have an incomplete pathologic response (pIR) since many similarly treated TNBC patients with identical stage and comparable residual cancer burden (RCB) demonstrate disparate clinical outcomes. Current methods fall short in predicting tumor recurrence and treatment resistance in the clinic. Supported by ample evidence in developmental, evolutionary, and cancer biology, we proposed that EGFR-K-RAS-SIAH pathway activation is a major driving force in TNBC and that its most downstream gatekeeper, SIAH, is a tumor-specific, therapy-responsive, and prognostic biomarker for patient risk stratification in TNBC. The persistent high expression of SIAH in residual tumors after neoadjuvant therapy reflects EGFR/K-RAS pathway activation (ON) and resistance to treatment. The loss of expression of SIAH in residual tumors reflects EGFR/K-RAS pathway inactivation (OFF) and response to treatment (i.e. treatment efficacy). Here we propose to establish the power of SIAH as a prognostic biomarker to differentiate high-risk from low-risk tumors and predict “real-time” therapy response, in both pre- and post-neoadjuvant (NACT/NST) setting. Breast cancer mortality rates in Hampton Roads Virginia and Richmond Virginia are among the worst in the U.S. and demonstrate a major cancer health disparity. Black patients in our region have a 60-70% higher mortality rate than white patients according to CDC/SEER databases. Using a large cohort of 525 racially diverse TNBC patients (246 black and 279 white patients) at Sentara Cancer Network, we conducted Kaplan-Meier survival analyses and detected a major racial disparity, with significantly lower survival among the Black/AA TNBC cohort, compared to national databases. We will expand this TNBC disparity study by using 2 large racially-diverse cohorts of 981 TNBC patients (459 Black and 493 white patients) from the Sentara Cancer Network and VCU Massey Cancer Center to analyze the prognosis of SIAH expression. By focusing on this tumor-driving EGFR-K-RAS-SIAH pathway, we aim to delineate the underlying molecular basis of treatment resistance and racial disparity in high-risk TNBC. Citation Format: Amy H. Tang, Richard A. Hoefer, Dasom Lee, Emily L. Breeding, Mary L. Guye, Janet S. Winston, Billur Samli, Jennifer Koblinski, Valentina Robila, Michael O. Idowu, Rick J. Jansen, Harry D. Bear. Early detection of cancer disparity and treatment resistance in TNBC. [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 5531.

Lung cancer is the second most diagnosed cancer. Lung cancer exhibits a sex difference, though the exact mechanisms behind this difference are not well understood. Men have a higher lifetime risk of developing lung cancer and often have more severe disease than women. There are thought to be multiple factors that contribute to this difference, including the environment, lifestyle, sex hormones, and differing immune responses. Mouse tumor models of lung, breast, colon, melanoma and kidney were used in immune competent or immune compromised mouse models treated with the chemotherapy ABT-263. The study of the immune response to lung tumors utilized ex vivo killing assays, multicolor flow cytometry, and clonogenic survival, as well as humanized mice and patient-derived xenografts. We observe a sex difference in two models of lung cancer in immunocompetent mice, but not in models of breast, colon, kidney, or melanoma. This difference is dependent on the innate immune system, specifically through natural killer cells and macrophages. This sex difference is lost in ovariectomized mice. Multicolor flow cytometry shows differences in innate immune cell populations in tumors and spleens in males and females. Preliminary data suggests the pro-apoptotic ligand TRAIL secreted from innate immune cells is more effective on cells exposed to female compared to ovariectomized female mouse serum. Reduced tumor growth in female mice can be further enhanced with ABT-263, and this effect requires NK cells. Patient-derived xenografts transplanted into female humanized mice grow more slowly than those transplanted into non-humanized mice, confirming the role of the immune system in modulating tumor growth. This research opens the potential for immune stimulating therapies, combined with ABT-263, as a novel treatment approach. Citation Format: Lauren A. May, Joseph W. Landry, Rebecca Martin, Howard Y. Li, Jennifer Koblinski, Paula D. Bos. Sex differences in the innate immune response to lung cancer [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 670.

Background: Lung cancer is the leading cause of cancer deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for ~80% of all lung cancers. Up to 69% of NSCLC are mutated for the tumor suppressor gene TP53, and of these, 84% are missense mutations, where wild-type (WT) TP53 tumor suppressor function is lost and in most cases, emergent cancer-promoting oncogenic activities are acquired. Due to the poor draggability of mutated p53 protein, the focus of therapeutics targeting NSCLC tumors with oncogenic missense mutant p53 has been on vulnerabilities that are not present in normal cells expressing WT p53. One such vulnerability involves oncogenic mutant p53-dependent attenuation of the oxidative stress response. The ability of cancer cells to survive oxidative stress depends on the regulated synthesis and activity of cellular antioxidants, including glutathione (GSH). Proteasome inhibitors (PIs) are known to induce cellular stress at multiple levels, including proteotoxic, autophagic, and oxidative stress, but the clinical utility of PIs has been limited by lack of efficacy in patients with solid tumors, including unselected lung cancer patients. Methods and Results: We now show bortezomib (BTZ) and other PIs are preferentially cytotoxic in oncogenic mutant p53-expressing lung cancer cells in culture. Furthermore, BTZ-treated oncogenic mutant p53-expressing cells display elevated levels of reactive oxygen species and BTZ’s cytotoxic effects were rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ cytotoxicity. Upon BTZ treatment of oncogenic mutant p53-expressing NSCLC cells, but not in WT p53 expressing NSCLC cells, we also observed the transcriptional induction of pro-apoptotic BH3-only BCL-2 family protein NOXA, consistent with an apoptotic mechanism of BTZ-induced cell death. Furthermore, in combination with BTZ, the use of BH3-mimetics such as venetoclax (ABT-199) or navitoclax (ABT-263) to inhibit the function of pro-survival proteins BCL-2/BCL-XL synergistically enhanced BTZ cytotoxicity, but only in oncogenic mutant p53-expressing NSCLC cells. In vivo mouse xenograft studies demonstrated that combination treatments of BTZ + venetoclax or BTZ + carboplatin (a standard of care chemotherapy for NSCLC) significantly attenuated oncogenic mutant p53-expressing tumor growth. Conclusions: Our data suggest that the preferential cytotoxicity of BTZ, and potentially other PIs, in oncogenic mutant p53-expressing NSCLC cells depends on the induction of toxic oxidative stress leading to NOXA-dependent apoptosis. Thus, combining PIs with drugs that limit antioxidant responses and/or sensitize cells to NOXA-induced apoptosis (e.g., BH3-mimetics) should synergistically and selectively kill NSCLC cells expressing oncogenic mutant p53 and open novel therapeutic avenues for the treatment of NSCLC. Citation Format: Kranthi Kumar Chougoni, Victoria L. Neely, Boxiao Ding, Eziafa Oduah, Khanh Nguyen, Bin Hu, Jennifer Koblinski, Bradford Windle, Hisashi Harada, Steven Grossman. Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors. [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 3950.

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heel” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

Background: Hepatocellular carcinoma (HCC) is a major clinical challenge because of increasing rates of incidence and mortality, and high resistance to standard radio- and chemotherapy. Identification of novel molecules regulating HCC facilitates development of targeted therapies having a lasting impact on HCC patient survival. The oncogene MDA-9/SDCBP promotes tumorigenesis and metastasis in many cancers. In The Cancer Genome Atlas (TCGA), 8% of HCC patients show amplification of MDA-9 gene. However, the role of MDA-9 in regulating HCC has not been well-studied. Objective: To unravel the function of MDA-9 in HCC using a transgenic mouse model with hepatocyte-specific overexpression of MDA-9 (Alb/MDA-9). Study design: Alb/MDA-9 mice were generated in B6/CBA background by using the mouse albumin promoter/enhancer element to drive human MDA-9 expression. N-nitrosodiethylamine (DEN)/phenobarbital (PB) was used for induction of HCC. Tumor immune profile was analyzed by Opal multiplex staining and flow cytometry. RNA-sequencing (RNA-seq) was performed to analyze MDA-9-mediated gene regulation. Results: At 18 months of age, none of the WT mice developed liver tumor, while one male and one female Alb/MDA-9 mice (n = 11) developed one isolated tumor each, suggesting that MDA-9 may not be a strong driver oncogene, rather it might promote HCC progression once tumor is initiated. Indeed, at 32 weeks after induction of HCC using DEN/PB, tumor burden in the livers of Alb/MDA-9 mice was significantly higher compared to WT. These tumors showed loss of hepatic architecture and hepatocyte ballooning, features of HCC. Increased staining for the proliferation marker PCNA, HCC markers AFP and cytokeratin, and angiogenesis marker CD31 in tumor sections, and significantly increased levels of serum liver enzymes, AST and ALT, were observed in Alb/MDA-9 mice vs WT. Macrophage-mediated inflammation plays a central role in HCC. Compared to WT, Alb/MDA-9 tumors showed marked infiltration of CD11b+ myeloid cells, FoxP3+ T regulatory cells and F4/80+ macrophages. RNA-seq in naïve WT and Alb/MDA-9 hepatocytes identified activation of signaling pathways associated with invasion, angiogenesis and inflammation. Conclusions: MDA-9 overexpression in hepatocytes promotes HCC by stimulating inflammation, angiogenesis and immune modulation. Studies are ongoing to obtain in-depth understanding of the molecular mechanisms by which MDA-9 exerts these pleiotropic effects. This study was supported by NIH/NCI Grant 1R01CA244993-01 (DS and PBF). Citation Format: Debashri Manna, Saranya Chidambaranathan Reghupaty, Rachel Gredler Mendoza, Maria Del Camarena, Mark A. Subler, Jennifer Koblinski, Rebecca Martin, Mikhail G. Dozmorov, Swadesh K. Das, Jolene J. Windle, Paul B. Fisher, Devanand Sarkar. Melanoma differentiation associated gene-9/syndecan binding protein (mda-9/sdcbp): A positive regulator of hepatocellular carcinoma [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 920.

Oral squamous cell carcinoma (OSCC) represents 3% of all cancer deaths in the U.S. and is ranked one of the top 10 cancers worldwide. The 5-year survival rate has remained at a low 50% for the past several decades, necessitating discovery of novel biomarkers of aggressive disease and therapeutic targets. As overexpression of urinary type plasminogen activator and receptor (uPA/R) in OSCC is associated with malignant progression and poor outcome, cell lines were generated with either overexpression (SCC25-uPAR+) or silencing (SCC25-uPAR-KD) of uPAR. As SCC25-uPAR+ tumors behaved more aggressively both in vitro and in vivo, comparative cDNA microarray analysis was used to identify additional genes that may be associated with aggressive tumors. Four members of the human tissue kallikrein family (KLK 5, 7, 8, and 10) were identified and real-time RT-PCR (qPCR) was used to verify and quantify gene expression. qPCR analysis revealed 2.8-, 5.3-, 4.0-, and 3.5-fold increases in gene expression for KLK5, 7, 8, and 10, respectively, in SCC25-uPAR+ versus SCC25-uPAR-KD. Immunohistochemical analysis demonstrated strong reactivity for KLKs 5, 7, 8 and 10 in both orthotopic murine tumors and human OSCC tissues. Control experiments show lack of reactivity against KLK3 (prostate specific antigen). These results demonstrate that kallikreins 5, 7, 8, and 10 are abundantly expressed in human OSCC and may be implicated in malignant progression.

Our previous studies suggest that the cysteine protease cathepsin B (catB) is involved in skeletal myoblast differentiation (myogenesis). To test this hypothesis, we examined the effect of trapping one of the two catB alleles on the ability of C2C12 cells to differentiate. During differentiation, catB gene-trapped C2C12 mouse myoblasts (RT-27) demonstrated a similar pattern of intracellular catB activity and protein expression compared to that observed in control C2C12 myoblasts and myoblasts trapped in a gene other than catB. However, compared to control myoblast cell lines, levels of catB activity and protein at each stage of RT-27 differentiation were reduced. The reductions in levels of catB were associated with reductions in several myogenic phenotypes including reduced levels of creatine phosphokinase activity and myosin heavy chain protein, two late biochemical markers of myogenesis, and reduced myotube size and extent of myotube formation over time. Comparable reductions were not observed for myogenin protein, an early biochemical marker of myogenesis, or in myokinase activity and catB related cathepsin L-type activity, two non-specific proteins. Finally, both control and catB gene-trapped myoblasts secreted active catB at pH 7.0. However levels of active pericellular/secreted catB were 50% lower in catB gene-trapped myoblasts. Collectively, these results support a functional link between catB expression and skeletal myogenesis and suggest a role for active pericellular/secreted catB in myoblast fusion.