Search

The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2,000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C; 80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the percentages of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D–deficient + NTCU (DN), 22.7% [P < 0.05 compared with vitamin D–sufficient +NTCU (SN)]; DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared with SN mice, DN mice exhibited a three-fold increase (P < 0.005) in circulating white blood cells (WBC), a 20% (P < 0.05) increase in IL6 levels, and a four-fold (P < 0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. Cancer Prev Res; 8(10); 895–904. ©2015 AACR.

Supplemental Table 1. Percent of Normal Airways, Atypia, Low-grade Dysplasia & High-grade Dysplasia Lesions Present in the Small Airways After NTCU Treatment

Activation of the epidermal growth factor receptor (EGFR) pathway is an early event in head and neck carcinogenesis. As a result, targeting EGFR for chemoprevention of head and neck squamous cell carcinomas (HNSCC) has received considerable attention. In the present study, we examined the impact of 1,25(OH)2D3, the active metabolite of the nutritional supplement vitamin D on the chemopreventive efficacy of the EGFR inhibitor, erlotinib, against HNSCC. Experimental studies were conducted in patient-derived xenografts (PDX) and the 4-nitroquinoline-1-oxide (4NQO) carcinogen-induced model of HNSCC. Short-term treatment (4 weeks) of PDX-bearing mice with 1,25(OH)2D3 and erlotinib resulted in significant inhibition of tumor growth. Noninvasive MRI enabled longitudinal monitoring of disease progression in the 4NQO model with 100% of control animals showing evidence of neoplastic lesions by 24 weeks. Among the experimental groups, animals treated with the combination regimen showed the greatest reduction in tumor incidence and volume (P < 0.05). Combination treatment was well tolerated and was not associated with any significant change in body weight. Histopathologic assessment revealed a significant reduction in the degree of dysplasia with combination treatment. Immunoblot analysis of whole tongue extracts showed downregulation of phospho-EGFR and phospho-Akt with the combination regimen. These results highlight the potential of 1,25(OH)2D3 to augment the efficacy of erlotinib against HNSCC. Further optimization of schedule and sequence of this combination regimen along with investigation into the activity of less calcemic analogues or dietary vitamin D is essential to fully realize the potential of this approach. Cancer Prev Res; 8(9); 765–76. ©2015 AACR.

Supplementary Fig. S1. MRI of 4NQO-induced tumor growth. Supplementary Fig. S2. MRI-based assessment of response to 1,25(OH)2D3 alone and in combination with erlotinib in the 4NQO model. Supplementary Fig. S3. Ex vivo assessment of response to combination treatment in the 4NQO model. Supplementary Fig. S4. Histopathologic assessment of response to combination treatment in the 4NQO model.

Supplemental Figure 1. Controls IHC stains

The optimization of linkers that connect fragments within drug binding sites represents an impediment in fragment-based drug discovery (FBDD). To improve our understand of the molecular factors that enable effective fragment linking, we have produced a series of compounds that bind to the ATP and allosteric sites of the EGFR kinase domain connected by two distinct linker structures. We find the linker is responsible for opposing impacts on potency against EGFR mutants, the most potent of which are active in human cancer cells. Comparison of X-ray cocrystal structures of active versus inactive molecules provide unique experimentally derived insights into linker design criteria such as how fragment flexibility and intermolecular interactions can serve compound design broadly in drug optimization.

Garlic, an Allium vegetable, contains rich flavonoids organosulfur compounds (OSCs) that have potent anticancer properties. The aim of the review is to provide an overview of the different types of garlic, their active compounds, and the potential anticancer benefits with a focus on antioxidant activity. Animal and cell line studies have provided convincing evidence that garlic and its organosulfur compounds inhibit carcinogenesis through a number of events including induction of apoptosis, inhibiting cellular proliferation, scavenging radical oxygen species (ROS), increasing the activities of enzymes such as glutathione S-transferase, and reducing tumor size. Epidemiological studies showed compelling evidence that garlic consumption is associated with decreased risk of colorectal cancer, but inconsistent evidence for stomach, breast, and prostate cancers. Studies also suggest that the presence and potency of garlic OSCs varies with respect to the preparation and form of garlic. Further epidemiological studies with information on garlic form consumed or preparation methods and molecular studies regarding its antioxidant mechanisms, such as increasing enzymatic and nonenzymatic antioxidants levels, are warranted.

Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

Background Vitamin D3 (VitD) deficiency is linked to increased incidence and worse survival in bladder cancer (BCa). In addition to cystectomy, patients are treated with cisplatin-based chemotherapy, however 30%-50% of patients do not benefit from this treatment. The effects of VitD deficiency on response to chemotherapy remain unknown. Methods To test effects of VitD supplementation on the response to cisplatin we analyzed patient serum VitD levels and correlated that with survival. In vivo, VitD deficient mice were treated with cisplatin, with or without pretreatment with the active VitD metabolite, 1,25 dihydroxyvitamin D3 (1,25D3 ). Lastly, using BCa cell lines, T24 and RT-112, the mechanism of action of 1,25D3 and cisplatin combination treatment was determined by apoptosis assays, as well as western blot and RT-PCR. Results In this study, we determined that low serum 25 hydroxyvitamin D3 (25D3 ) levels was significantly associated with worse response to cisplatin. Pretreating deficient mice with 1,25D3 , reduced tumor volume compared to cisplatin monotherapy. In vitro, 1,25D3 pretreatment increased the apoptotic response to cisplatin. 1,25D3 pretreatment increased expression of TAp73 and its pro-apoptotic targets, in a VDR dependent manner. VDR and its transcriptional targets were induced after 1,25D3 treatment and further increased after the combination of 1,25D3 and cisplatin in a TAp73 dependent manner. Conclusions Our data suggest that VitD deficiency could be a biomarker for poor response to cisplatin, and pretreating with VitD can increase the apoptotic response to cisplatin through VDR and TAp73 signaling crosstalk.

The anticancer activity of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol) has been widely reported in preclinical models. However, systematic investigation into the chemopreventive potential of calcitriol against the spectrum of oral carcinogenesis has not been performed. To address this gap in knowledge, we conducted a preclinical prevention trial of calcitriol in the 4-nitroquinoline-1-oxide (4NQO) oral carcinogenesis model. C57BL/6 mice were exposed to the carcinogen 4NQO in drinking water for 16 weeks and randomized to control (4NQO only) or calcitriol arms. Calcitriol (0.1 μg i.p, Monday, Wednesday, and Friday) was administered for (i) 16 weeks concurrently with 4NQO exposure, (ii) 10 weeks post completion of 4NQO exposure, and, (iii) a period of 26 weeks concurrent with and following 4NQO exposure. Longitudinal magnetic resonance imaging (MRI) was performed to monitor disease progression until end point (week 26). Correlative histopathology of tongue sections was performed to determine incidence and multiplicity of oral dysplastic lesions and squamous cell carcinomas (SCC). Vitamin D metabolites and calcium were measured in the serum using liquid chromatography-mass spectrometry (LC–MS/MS) and colorimetric assay, respectively. Renal CYP24A1 (24-hydroxylase) and CYP27B1 (1α-hydroxylase) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Immunostaining of tongue sections for vitamin D receptor (VDR), CYP24A1, and Ki67 was also performed. Non-invasive MRI enabled longitudinal assessment of lesions in the oral cavity. Calcitriol administered concurrently with 4NQO for 16 weeks significantly (P

Advanced lung cancer patients that present with activating mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). While initially effective, all patients eventually develop therapeutic resistance and experience disease progression. Strategies to prevent EGFR TKI resistance are needed to improve patient outcomes. We chronically exposed H1975 cells (EGFR-L858R/T790M) to EGFR TKI osimertinib to study emergence of resistance. H1975 cells that were expanded under drug treatment (designated H1975OR) acquired an EMT phenotype and were re-sensitized to EGFR TKI upon prolonged drug withdrawal. These features led us to classify H1975OR as a model of drug tolerance rather than a model of stable drug resistance. Bulk RNA-sequencing revealed significant dysregulation of chromatin modifying genes in H1975OR. Bromodomain containing protein 9 (BRD9) was among the set of significantly upregulated chromatin regulators and was selected for further investigation as a mediator of drug tolerance. Although BRD9 is known to control stemness and EMT, its role in promoting EMT-associated TKI resistance is unknown. To test the contribution of BRD9 to EGFR TKI tolerance, pharmacological inhibition of BRD9 by the selective inhibitor, I-BRD9 significantly increased sensitivity to TKI in models with EMT phenotypes (IC50 reduced 3-4 fold). In contrast, I-BRD9 did not affect TKI sensitivity in two models where EGFR TKI resistance was genetically fixed. To gain mechanistic insights, H1975OR cells were treated with osimertinib +/-I-BRD9 and subjected to RNA-sequencing. Combination of osimertinib with I-BRD9 resulted in a significant decrease in EMT-related genes, including MMP9, Zeb2, PDGFRb and IL6. Further, use of I-BRD9 in these models diminished the mesenchymal phenotype, as measured in cell invasion assays. Genetic knockdown of BRD9 via shRNA phenocopied effects of I-BRD9 treatment, supporting BRD9 as the therapeutic target of I-BRD9 in our cell line models. To further establish a role for BRD9 in the emergence of drug tolerant cells, we exposed treatment naïve H1975 cells to EGFR TKI ± I-BRD9. I-BRD9 significantly decreased the size of the EGFR TKI tolerant population. In time course studies, I-BRD9 also delayed onset of TKI resistance. In conclusion, our data identifies BRD9 as a novel mediator of EMT-associated EGFR-TKI tolerance in EGFR-mutant lung cancer. Our data further implicates BRD9 inhibition as a novel strategy to delay the emergence of drug tolerant cells that eventually give rise to stable drug resistance. This work was supported by the Roswell Park Alliance Foundation and National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Genomics and Bioinformatics Shared Resources. Citation Format: Hannah Calkins, Tatiana Shaurova, David W. Goodrich, Mukund Seshadri, Candace S. Johnson, Pamela A. Hershberger. BRD9 inhibition overcomes epithelial to mesenchymal transition (EMT)-associated tyrosine kinase inhibitor (TKI) tolerance in epidermal growth factor receptor (EGFR) mutant lung cancer [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 1093.

Garlic, an Allium vegetable, contains rich flavonoids organosulfur compounds (OSCs) that have potent anticancer properties. The aim of the review is to provide an overview of the different types of garlic, their active compounds, and the potential anticancer benefits with a focus on antioxidant activity. Animal and cell line studies have provided convincing evidence that garlic and its organosulfur compounds inhibit carcinogenesis through a number of events including induction of apoptosis, inhibiting cellular proliferation, scavenging radical oxygen species (ROS), increasing the activities of enzymes such as glutathione S-transferase, and reducing tumor size. Epidemiological studies showed compelling evidence that garlic consumption is associated with decreased risk of colorectal cancer, but inconsistent evidence for stomach, breast, and prostate cancers. Studies also suggest that the presence and potency of garlic OSCs varies with respect to the preparation and form of garlic. Further epidemiological studies with information on garlic form consumed or preparation methods and molecular studies regarding its antioxidant mechanisms, such as increasing enzymatic and nonenzymatic antioxidants levels, are warranted.

EGFR tyrosine kinase inhibitors (EGFR TKIs) are the standard of care treatment for patients with EGFR-mutant lung adenocarcinoma (LUAD). Although initially effective, EGFR TKIs are not curative. Disease inevitably relapses due to acquired drug resistance. We hypothesized that vitamin D metabolites could be used with EGFR TKIs to prevent therapeutic failure. To test this idea, we investigated the link between serum 25-hydroxyvitamin D3 (25(OH)D3) and progression-free survival (PFS) in patients with EGFR-mutant LUAD that received EGFR TKIs (erlotinib n = 20 and afatinib n = 1). Patients who were 25(OH)D3-sufficient experienced significantly longer benefit from EGFR TKI therapy (mean 14.5 months) than those with 25(OH)D3 insufficiency (mean 10.6 months, p = 0.026). In contrast, 25(OH)D3 had no prognostic value in patients with KRAS-mutant LUAD that received cytotoxic chemotherapy. To gain mechanistic insights, we tested 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity in vitro. 1,25(OH)2D3 promoted epithelial differentiation and restored EGFR TKI sensitivity in models of EGFR TKI resistance that were associated with epithelial&ndash, mesenchymal transition (EMT). 1,25(OH)2D3 was ineffective in a non-EMT model of resistance. We conclude that vitamin D sufficiency portends increased PFS among EGFR-mutant LUAD patients that receive EGFR TKIs, and that vitamin D signaling maintains drug efficacy in this specific patient subset by opposing EMT.

Somatic alterations in the epidermal growth factor receptor gene (EGFR) result in aberrant activation of kinase signaling and occur in ∼15% of non-small cell lung cancers (NSCLC). Patients diagnosed with EGFR-mutant NSCLC have good initial clinical response to EGFR tyrosine kinase inhibitors (EGFR TKIs), yet tumor recurrence is common and quick to develop. Mechanisms of acquired resistance to EGFR TKIs have been studied extensively over the past decade. Great progress has been made in understanding two major routes of therapeutic failure: additional genomic alterations in the EGFR gene and activation of alternative kinase signaling (so-called “bypass activation”). Several pharmacological agents aimed at overcoming these modes of EGFR TKI resistance are FDA-approved or under clinical development. Phenotypic transformation, a less common and less well understood mechanism of EGFR TKI resistance is yet to be addressed in the clinic. In the context of acquired EGFR TKI resistance, phenotypic transformation encompasses epithelial to mesenchymal transition (EMT), transformation of adenocarcinoma of the lung (LUAD) to squamous cell carcinoma (SCC) or small cell lung cancer (SCLC). SCLC transformation, or neuroendocrine differentiation, has been linked to inactivation of TP53 and RB1 signaling. However, the exact mechanism that permits lineage switching needs further investigation. Recent reports indicate that LUAD and SCLC have a common cell of origin, and that trans-differentiation occurs under the right conditions. Options for therapeutic targeting of EGFR-mutant SCLC are limited currently to conventional genotoxic chemotherapy. Similarly, the basis of EMT-associated resistance is not clear. EMT is a complex process that can be characterized by a spectrum of intermediate states with diverse expression of epithelial and mesenchymal factors. In the context of acquired resistance to EGFR TKIs, EMT frequently co-occurs with bypass activation, making it challenging to determine the exact contribution of EMT to therapeutic failure. Reversibility of EMT-associated resistance points toward its epigenetic origin, with additional adjustments, such as genetic alterations and bypass activation, occurring later during disease progression. This review will discuss the mechanistic basis for EGFR TKI resistance linked to phenotypic transformation, as well as challenges and opportunities in addressing this type of targeted therapy resistance in EGFR-mutant NSCLC.

Despite the recognized link between aging and cancer, most preclinical studies in experimental tumor models are conducted with 6- to 8-wk-old rodents. The goal of the present study was to examine the impact of age on tumor incidence, growth, and microenvironmental characteristics in mouse models of head and neck squamous cell carcinoma (HNSCC). Experimental studies were conducted with the 4-nitroquinoline-oxide (4NQO) oral carcinogenesis model and orthotopic FaDu HNSCC xenografts, established in young (7 to 12 wk of age) and old (65 to 70 wk of age) female C57BL/6 mice ( n = 44; 4NQO model) and severe combined immunodeficient mice ( n = 13; HNSCC xenografts). Noninvasive whole body magnetic resonance imaging revealed increased subcutaneous and visceral fat in aging animals of both strains. On histologic examination, a higher incidence ( P < 0.001) of severe dysplasia/invasive squamous cell carcinoma was observed in old mice (92%) as compared with young mice (69%). Old C57BL/6 mice exposed to 4NQO exhibited increased incidence of oral and extraoral (peritoneal masses) neoplasms (42%) versus their young counterparts ( P < 0.05). The incidence of extraoral neoplasms was significantly lower (16%) in the younger cohort. Interestingly, no difference in growth rate and oxygen saturation was observed between orthotopic FaDu xenografts established in old and young severe combined immunodeficient mice. Our observations suggest that host age may have an impact on the growth kinetics and progression of HNSCC in the immunocompetent 4NQO model. Further investigation into the impact of aging on tumor response to preventive and therapeutic intervention is warranted.

Recent studies highlight the importance of the RB1 tumor suppressor as a target for cancer therapy. Canonically, RB1 regulates cell cycle progression and represents the downstream target for cyclin-dependent kinase (CDK) 4/6 inhibitors that are in clinical use. However, newly discovered features of the RB1 pathway suggest new therapeutic strategies to counter resistance and improve precision medicine. These therapeutic strategies include deepening cell cycle exit with CDK4/6 inhibitor combinations, selectively targeting tumors that have lost RB1, and expanding therapeutic index by mitigating therapy-associated adverse effects. In addition, RB1 impacts immunological features of tumors and the microenvironment that can enhance sensitivity to immunotherapy. Lastly, RB1 specifies epigenetically determined cell lineage states that are disrupted during therapy resistance and could be re-installed through the direct use of epigenetic therapies. Thus, new opportunities are emerging to improve cancer therapy by exploiting the RB1 pathway.

Radiation therapy (RT) is an integral component of the standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While advances in radiation delivery methods have led to improved outcomes, radioresistance poses a significant therapeutic challenge and a major cause of poor prognosis in this patient population. To date, a majority of studies investigating mechanisms of radioresistance have employed in vitro monolayer cell culture models that lack the heterogeneity and cell-extracellular matrix interactions. To overcome this limitation, we established and credentialed a panel of 3D patient-derived organoid (PDO) models of HNSCC to identify molecular signatures and signaling pathways that contribute to radiation resistance. A total of 7 PDOs were established from surgical HNSCC specimens and credentialed using a combination of histologic/immunohistochemical evaluation (H&E, cytokeratin, p16), genotyping (HPV), whole exome sequencing (WES) and RNA sequencing analysis. Radiation response of the PDO panel was also examined and assessed for concordance with patient response and PDOs were ranked based on their radiosensitivity and bioinformatic analysis was performed on radiosensitive and radioresistance PDOs to identify molecular pathways associated with resistance. All PDOs retained the histology, p16/HPV status and mutational landscape (p53, HLA-A) of donor patient tissues. Radiation response of PDOs showed excellent (100%) concordance with patient responses (n=5 with history of radiation treatment). RNA-seq revealed identified pathways associated with epithelial mesenchymal transition (IGFBP2, TIMP1, TIMP2, LAMA3, LOXL1), NOTCH signaling (NOTCH1, HES1, FZD1, FZD5, DTX1, NOTCH3, FZD7), E2F targets and G2M checkpoint signaling (MCM2, MCM7, MCM4, PCNA) and the p53 pathway (SFN, NDRG1, ALOX15B). Collectively, our findings illustrate the translational utility of PDOs as a robust platform in identifying potential therapeutic targets that could overcome radiation resistance in HNSCC. Ongoing studies are evaluating the potential of PDOs to serve as a screening platform to identify radiosensitizers through repurposing of FDA-approved drugs to facilitate rapid clinical translation. Citation Format: Vui King Vincent-Chong, Christian Gluck, Pamela A. Hershberger, Satrajit Sinha, Mukund Seshadri. Deciphering radiation resistance in head and neck cancer using patient derived organoids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-061.

Patient-derived organoids (PDOs) are considered to be a cost-effective, high-throughput preclinical platform for studying cancer biology and evaluating novel therapeutics. While PDOs have been established and characterized for breast, prostate, colon, and pancreatic cancers, PDO models of head and neck squamous cell carcinomas (HNSCC) are limited. To address this paucity, in the present study, we established a panel of PDO models of HNSCC. Five HNSCC samples (3 larynx, 1 tongue, 1 parotid) were procured from donor patients following informed consent and processed to establish organoids. Histologic (H&E) and immunohistochemical evaluation (p16, cytokeratin) was performed along with assessment of PDO response to the alkylating agent, cisplatin, and two tyrosine kinase inhibitors, erlotinib and afatinib. Histologic analyses of established organoids revealed nested cells of squamous cell carcinoma with high N/C ratio similar to donor surgical tumor tissue. Immunohistochemistry revealed strong pan cytokeratin and absence of p16 staining in all samples. Paired PDO and PDX models also successfully retained the squamous histology. Dose-response curves revealed substantial variation in therapeutic profiles of the three agents across the organoid panel. While the IC50 of cisplatin was comparable across the larynx organoids, 2 larynx organoids were sensitive to the two TKIs while one was resistant, exhibiting ~5-fold higher IC50 values. The tongue organoid was relatively sensitive to all agents while the parotid SCC organoid was sensitive to EGFR TKIs but relatively resistant to cisplatin. Collectively, these results demonstrate the ability of organoid models to recapitulate the histologic architecture and response heterogeneity of human HNSCC. Investigation into the molecular profiles of our PDO panel is under way in our laboratory and will enable further development of organoids as a robust platform for evaluation of precision therapeutics against HNSCC. Citation Format: Vincent-Chong Vui King, Erin C. Tracy, Heinz Baumann, Pamela A Hershberger, Mukund Seshadri. Patient-derived organoids recapitulate response heterogeneity in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A22.

Calcitriol, the active metabolite of vitamin D, has been widely studied for its preventive and therapeutic activity against several cancers including oral squamous cell carcinoma (OSCC). However, the impact of dietary vitamin D supplementation on initiation and progression of OSCC is unclear. To address this gap in knowledge, we conducted preclinical trials using the 4-nitroquinoline-1-oxide (4NQO) carcinogen model of oral carcinogenesis. Female C57BL/6 mice were maintained on one of three vitamin D diets [ 25 IU, 100 IU, 10,000 IU] and exposed to 4NQO in drinking water for 16 weeks followed by regular water for 10 weeks. Body weight measurements obtained through the study duration did not reveal any differences between the three diets. Animals on 100 IU diet showed lower incidence of high-grade dysplasia/OSCC and higher CD3+ T cells compared to animals on 25 IU and 10000 IU diets. Serum 25(OH)D(3) levels were highest in animals on 10000 IU diet at week 0 (prior to carcinogen exposure) but showed ~50% reduction at week 26. Histologic evaluation revealed highest incidence of OSCC in animals maintained on 10000 IU diet. Animals on 100 IU and 10000 IU diets showed higher vitamin D receptor (VDR) and CYP24A1 immunostaining in high-grade dysplastic lesions and OSCC compared to normal tongue. Validation studies performed in a 4NQO-derived OSCC model showed that short-term treatment of animals on a 25 IU diet with calcitriol significantly inhibited tumor growth compared to controls but did not affect tumor growth in animals on reference diet (1000 IU). Collectively, our results highlight the complex dynamics between vitamin D status and oral carcinogenesis. Our observations also suggest that therapeutic benefits of short-term calcitriol treatment may be more pronounced in vitamin D deficient hosts.

Background Vitamin D3 (VitD) deficiency is linked to increased incidence and worse survival in bladder cancer (BCa). In addition to cystectomy, patients are treated with cisplatin‐based chemotherapy, however 30%‐50% of patients do not benefit from this treatment. The effects of VitD deficiency on response to chemotherapy remain unknown. Methods To test effects of VitD supplementation on the response to cisplatin we analyzed patient serum VitD levels and correlated that with survival. In vivo, VitD deficient mice were treated with cisplatin, with or without pretreatment with the active VitD metabolite, 1,25 dihydroxyvitamin D3 (1,25D3). Lastly, using BCa cell lines, T24 and RT‐112, the mechanism of action of 1,25D3 and cisplatin combination treatment was determined by apoptosis assays, as well as western blot and RT‐PCR. Results In this study, we determined that low serum 25 hydroxyvitamin D3 (25D3) levels was significantly associated with worse response to cisplatin. Pretreating deficient mice with 1,25D3, reduced tumor volume compared to cisplatin monotherapy. In vitro, 1,25D3 pretreatment increased the apoptotic response to cisplatin. 1,25D3 pretreatment increased expression of TAp73 and its pro‐apoptotic targets, in a VDR dependent manner. VDR and its transcriptional targets were induced after 1,25D3 treatment and further increased after the combination of 1,25D3 and cisplatin in a TAp73 dependent manner. Conclusions Our data suggest that VitD deficiency could be a biomarker for poor response to cisplatin, and pretreating with VitD can increase the apoptotic response to cisplatin through VDR and TAp73 signaling crosstalk.

Mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor ( EGFR) gene drives the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR TKIs), including erlotinib and afatinib, are initially effective in treating EGFR mutant nonsmall cell lung cancer (NSCLC). However, drug resistance quickly develops due to several mechanisms, including induction of the epithelial-mesenchymal transition (EMT). No effective therapies are currently available for patients who develop EMT-associated EGFR TKI resistance. 1,25-Dihydroxyvitamin D3 (1,25D3) promotes epithelial differentiation and inhibits growth of NSCLC cells. 1,25D3 thus represents a promising agent for the treatment of EMT-associated EGFR TKI resistance. However, 1,25D3 induces the expression of 24-hydroxylase (24OHase), which decreases 1,25D3 activity. CTA091, a potent and selective 24OHase inhibitor, has been developed to attenuate this adverse effect. CTA091 also suppresses renal 24OHase activity and so may promote hypercalcemia. To exploit favorable effects of 1,25D3 plus CTA091 in tumor cells while avoiding problematic systemic effects of 24OHase inhibition, we developed EGFR-targeted, liposomal nanoparticles (EGFR-LP) to offer tumor-targeted co-delivery of 1,25D3 and CTA091. We then established an EMT-associated model of EGFR TKI resistance, and showed that such nanoparticles improved cellular uptake of 1,25D3 and CTA091, drove pro-epithelial signaling by upregulating E-cadherin ( CDH1), and significantly inhibited the growth of EGFR TKI resistant cells. Our results demonstrated that the delivery of vitamin D-based drug payloads via tumor-targeted EGFR-LP has promise as a new therapy for EFGR TKI resistant lung cancer. Future studies will focus on in vivo evaluation of biological activity, therapeutic benefits, and systemic toxicity prior to clinical translation.

Vitamin D metabolites exhibit chemopreventive, antiproliferative, and antimetastatic activity in preclinical models of lung cancer. Besides having direct actions on tumor cells, vitamin D may control lung cancer by favorably altering the lung tumor microenvironment and immune function. Several factors are likely to modulate vitamin D activity in lung cancer patients including active cigarette smoke exposure, somatic variation in vitamin D pathway genes, and molecular heterogeneity of lung tumors. Promising data exist regarding vitamin D actions in individuals who have chronic obstructive pulmonary disease and are at elevated risk for lung cancer. However, vitamin D-containing therapies have yet to demonstrate significant activity in unselected, advanced lung cancer patients. Future trials may benefit from a personalized medicine approach wherein the molecular profile of a patient's lung tumor and a patient's vitamin D status are used to identify individuals who are more likely to benefit from vitamin D supplementation.

Epidemiologic studies implicate vitamin D status as a factor that influences growth of EGFR mutant lung cancers. However, laboratory based evidence of the biological effect of vitamin D in this disease is lacking. To fill this knowledge gap, we determined vitamin D receptor (VDR) expression in human lung tumors using a tissue microarray constructed of lung cancer cases from never-smokers (where EGFR gene mutations are prevalent). Nuclear VDR was detected in 19/19 EGFR mutant tumors. Expression tended to be higher in tumors with EGFR exon 19 deletions than those with EGFR L858R mutations. To study anti-proliferative activity and signaling, EGFR mutant lung cancer cells were treated with the circulating metabolite of vitamin D, 25-hydroxyvitamin D3 (25D3). 25D3 inhibited clonogenic growth in a dose-dependent manner. CYP27B1 encodes the 1α-hydroxylase (1αOHase) that converts 25D3 to the active metabolite, 1,25-dihydroxyvitamin D3 (1,25D3). Studies employing VDR siRNA, CYP27B1 zinc finger nucleases, and pharmacologic inhibitors of the vitamin D pathway indicate that 25D3 regulates gene expression in a VDR-dependent manner but does not strictly require 1αOHase-mediated conversion of 25D3 to 1,25D3. To determine the effects of modulating serum 25D3 levels on growth of EGFR mutant lung tumor xenografts, mice were fed diets containing 100 or 10,000 IU vitamin D3/kg. High dietary vitamin D3 intake resulted in elevated serum 25D3 and significant inhibition of tumor growth. No toxic effects of supplementation were observed. These results identify EGFR mutant lung cancer as a vitamin D-responsive disease and diet-derived 25D3 as a direct VDR agonist and therapeutic agent.

The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2,000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C; 80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the percentages of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D–deficient + NTCU (DN), 22.7% [P < 0.05 compared with vitamin D–sufficient +NTCU (SN)]; DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared with SN mice, DN mice exhibited a three-fold increase (P < 0.005) in circulating white blood cells (WBC), a 20% (P < 0.05) increase in IL6 levels, and a four-fold (P < 0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. Cancer Prev Res; 8(10); 895–904. ©2015 AACR.

Benzaldehyde dimethane sulfonate (BEN, DMS612, NSC281612) is a bifunctional alkylating agent currently in clinical trials. We previously characterized the degradation products of BEN in plasma and blood. The conversion of BEN to its carboxylic acid analogue (BA) in whole blood, but not plasma, suggests that an enzyme in RBCs may be responsible for this conversion. BEN conversion to BA was observed in renal carcinoma cells and appeared to correlate with IC₅₀. To better understand the pharmacology of BEN, we aimed to evaluate the metabolism and enzymes potentially responsible for the conversion of BEN to BA.Human red blood cells (RBC) were used to characterize kinetics and susceptibility to enzyme-specific inhibitors. Recombinant enzymes were used to confirm metabolism of BEN to BA. Analytes were quantitated with established LC-MS/MS methods.Average apparent Vmax and Km were 68 ng/mL min(-1) [10% RBC](-1) and 373 ng/mL, respectively. The conversion of BEN to BA in RBC was not inhibited by carbon monoxide, nitrogen gas, or menadione, an inhibitor of aldehyde oxidase. The conversion was inhibited by disulfiram, an inhibitor of ALDH. Of available ALDH isoforms ALDH1A1, ALDH3A1, ALDH2, and ALDH5A1, only ALDH1A1 converted BEN to BA.The activating conversion of BEN to BA is mediated not by CYP450 enzymes or aldehyde oxidase, but by ALDH1A1. This enzyme, a potential stem cell marker, may be a candidate biomarker for clinical activity of BEN.

Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

Veliparib (ABT-888) is an oral PARP inhibitor expected to increase gemcitabine activity. This phase I determined the maximal tolerable dose (MTD), dose-limiting toxicities (DLT), antitumor activity, pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib combined with gemcitabine.Patients with advanced solid tumors received veliparib (10-40-mg PO BID) on chemotherapy weeks with gemcitabine 500-750-mg/mEleven patients were enrolled on the 28-day schedule. The 28-day schedule was considered intolerable and amended to a 21-day schedule, with 20 patients enrolled. Grade ≥ 3 adverse events were myelosuppression-related. The MTD was determined to be 750-mg/mGemcitabine at 750-mg/m

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts anti-proliferative activity by binding to the vitamin D receptor (VDR) and regulating gene expression. We previously reported that non-small cell lung cancer (NSCLC) cells which harbor epidermal growth factor receptor (EGFR) mutations display elevated VDR expression (VDRhigh) and are vitamin D-sensitive. Conversely, those with K-ras mutations are VDRlow and vitamin D-refractory. Because EGFR mutations are found predominately in NSCLC cells with an epithelial phenotype and K-ras mutations are more common in cells with a mesenchymal phenotype, we investigated the relationship between vitamin D signaling capacity and the epithelial mesenchymal transition (EMT). Using NSCLC cell lines and publically available lung cancer cell line microarray data, we identified a relationship between VDR expression, 1,25(OH)2D3 sensitivity, and EMT phenotype. Further, we discovered that 1,25(OH)2D3 induces E-cadherin and decreases EMT-related molecules SNAIL, ZEB1, and vimentin in NSCLC cells. 1,25(OH)2D3-mediated changes in gene expression are associated with a significant decrease in cell migration and maintenance of epithelial morphology. These data indicate that 1,25(OH)2D3 opposes EMT in NSCLC cells. Because EMT is associated with increased migration, invasion, and chemoresistance, our data imply that 1,25(OH)2D3 may prevent lung cancer progression in a molecularly defined subset of NSCLC patients.

Background Cetuximab has demonstrated improved efficacy in combination with chemotherapy and radiotherapy. We evaluated the integration of cetuximab in the combined modality treatment of stage III non-small cell lung cancer (NSCLC). Methods Patients with surgically unresectable stage IIIA or IIIB NSCLC were treated with chest radiotherapy, 73.5 Gy (with lung and tissue heterogeneity corrections) in 35 fractions/7 weeks, once daily (63 Gy without heterogeneity corrections). Cetuximab was given weekly during radiotherapy and continued during consolidation therapy with carboplatin and paclitaxel up to a maximum of 26 weekly doses. The primary endpoint was overall survival. Baseline tumor tissue was analyzed for EGFR by fluorescence in situ hybridization (FISH). Results Forty patients were enrolled in this phase II study. The median overall survival was 19.4 months and the median progression-free survival 9.3 months. The best overall response rate in 31 evaluable patients was 67%. No grade 3 or 4 esophagitis was observed. Three patients experienced grade 3 rash; 16 patients (69%) developed grade 3/4 neutropenia during consolidation therapy. One patient died of pneumonitis, possibly related to cetuximab. EGFR gene copy number on baseline tumor tissues, analyzed by FISH, was not predictive of efficacy outcomes. Conclusions The addition of cetuximab to chest radiotherapy and consolidation chemotherapy was tolerated well and had modest efficacy in stage III NSCLC. Taken together with the lower incidence of esophagitis, our results support evaluation of targeted agents instead of chemotherapy with concurrent radiotherapy in this setting.

The active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in regulating calcium homeostasis and bone mineralization. 1,25(OH)2D3 also modulates cellular proliferation and differentiation in a variety of cell types. 24-Hydroxylase, encoded by the CYP24A1 gene, is the key enzyme which converts 1,25(OH)2D3 to less active calcitroic acid. Nearly all cell types express 24-hydroxylase, the highest activity being observed in the kidney. There is increasing evidence linking the incidence and prognosis of certain cancers to low serum 25(OH)D3 levels and high expression of vitamin D 24-hydroxylase, supporting the idea that elevated CYP24A1 expression may stimulate degradation of vitamin D metabolites including 25(OH)D3 and 1,25(OH)2D3. The over expression of CYP24A1 in cancer cells may be a factor affecting 1,25(OH)2D3 bioavailability and anti-proliferative activity pre-clinically and clinically. The combination of 1,25(OH)2D3 with CYP24A1 inhibitors enhances 1,25(OH)2D3 mediated signaling and anti-proliferative effects and may be useful in overcoming effects of aberrant CYP24A1 expression. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

We previously demonstrated that non-small cell lung cancer (NSCLC) cells and primary human lung tumors aberrantly express the vitamin D3-catabolizing enzyme, CYP24, and that CYP24 restricts transcriptional regulation and growth control by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in NSCLC cells. To ascertain the basis for CYP24 dysregulation, we assembled a panel of cell lines that represent distinct molecular classes of lung cancer: cell lines were selected which harbored mutually exclusive mutations in either the K-ras or the Epidermal Growth Factor Receptor (EGFR) genes. We observed that K-ras mutant lines displayed a basal vitamin D receptor (VDR)(low)CYP24(high) phenotype, whereas EGFR mutant lines had a VDR(high)CYP24(low) phenotype. A mutation-associated difference in CYP24 expression was also observed in clinical specimens. Specifically, K-ras mutation was associated with a median 4.2-fold increase in CYP24 mRNA expression (p=4.8×10(-7)) compared to EGFR mutation in a series of 147 primary lung adenocarcinoma cases. Because of their differential basal expression of VDR and CYP24, we hypothesized that NSCLC cells with an EGFR mutation would be more responsive to 1,25(OH)2D3 treatment than those with a K-ras mutation. To test this, we measured the ability of 1,25(OH)2D3 to increase reporter gene activity, induce transcription of endogenous target genes, and suppress colony formation. In each assay, the extent of 1,25(OH)2D3 response was greater in EGFR mutation-positive HCC827 and H1975 cells than in K-ras mutation-positive A549 and 128.88T cells. We subsequently examined the effect of combining 1,25(OH)2D3 with erlotinib, which is used clinically in the treatment of EGFR mutation-positive NSCLC. 1,25(OH)2D3/erlotinib combination resulted in significantly greater growth inhibition than either single agent in both the erlotinib-sensitive HCC827 cell line and the erlotinib-resistant H1975 cell line. These data are the first to suggest that EGFR mutations may identify a lung cancer subset which remains responsive to and is likely to benefit from 1,25(OH)2D3 administration. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Ligand structure can affect the activation of nuclear receptors, such as estrogen receptors (ERs), and their control of signaling pathways for cellular responses including death and differentiation. We hypothesized that distinct biological functions of similar estradiol (E2) analogs could be identified by integrating gene expression patterns obtained from human tumor cell lines with receptor binding and functional data for the purpose of developing compounds for treatment of a variety of diseases. We compared the estrogen receptor subtype selectivity and impact on signaling pathways for three distinct, but structurally similar, analogs of E2. Modifications in the core structure of E2led to pronounced changes in subtype selectivity for estrogen receptors, ER-α or ER-β, along with varying degrees of ER dimerization and activation. While all three E2analogs are predominantly ER-β agonists, the cell growth inhibitory activity commonly associated with this class of compounds was detected for only two of the analogs and might be explained by a ligand-specific pattern of gene transcription. Microarray studies using three different human tumor cell lines demonstrated that the analogs distinctly affect the transcription of genes in signaling pathways for chromosome replication, cell death, and oligodendrocyte progenitor cell differentiation. That the E2analogs could lower tumor cell viability and stimulate neuronal differentiation confirmed that gene expression data could accurately distinguish biological activity of the E2analogs. The findings reported here confirm that cellular responses can be regulated by making key structural alterations to the core structure of endogenous ER ligands.

Background E-cigarettes or electronic nicotine delivery systems (ENDS) are designed to deliver nicotine-containing aerosol via inhalation. Little is known about the health effects of flavoured ENDS aerosol when inhaled. Methods Aerosol from ENDS was generated using a smoking machine. Various types of ENDS devices or a tank system prefilled with liquids of different flavours, nicotine carrier, variable nicotine concentrations and with modified battery output voltage were tested. A convenience sample of commercial fluids with flavour names of tobacco, pina colada, menthol, coffee and strawberry were used. Flavouring chemicals were identified using gas chromatography/mass spectrometry. H292 human bronchial epithelial cells were directly exposed to 55 puffs of freshly generated ENDS aerosol, tobacco smoke or air (controls) using an air–liquid interface system and the Health Canada intense smoking protocol. The following in vitro toxicological effects were assessed: (1) cell viability, (2) metabolic activity and (3) release of inflammatory mediators (cytokines). Results Exposure to ENDS aerosol resulted in decreased metabolic activity and cell viability and increased release of interleukin (IL)-1β, IL-6, IL-10, CXCL1, CXCL2 and CXCL10 compared to air controls. Cell viability and metabolic activity were more adversely affected by conventional cigarettes than most tested ENDS products. Product type, battery output voltage and flavours significantly affected toxicity of ENDS aerosol, with a strawberry-flavoured product being the most cytotoxic. Conclusions Our data suggest that characteristics of ENDS products, including flavours, may induce inhalation toxicity. Therefore, ENDS users should use the products with caution until more comprehensive studies are performed.

Human lung tumors aberrantly express the 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 )-catabolizing enzyme, CYP24. We hypothesized that CYP24 reduces 1,25(OH) 2 D 3 -mediated transcription and allows lung cancer cells to escape its growth-inhibitory action. To test this, H292 lung cancer cells and the CYP24-selective inhibitor CTA091 were utilized. In H292 cells, CTA091 reduces 1,25(OH) 2 D 3 catabolism, significantly increases 1,25(OH) 2 D 3 -mediated growth inhibition, and increases 1,25(OH) 2 D 3 effects on induced and repressed genes in gene expression profiling studies. Pathway mapping of repressed genes uncovered cell cycle as a predominant 1,25(OH) 2 D 3 target. In H292 cells, 1,25(OH) 2 D 3 significantly decreases cyclin E2 levels and induces G 0 /G 1 arrest. A broader set of cyclins is down-regulated when 1,25(OH) 2 D 3 is combined with CTA091, and cell cycle arrest further increases. Effects of CTA091 on 1,25(OH) 2 D 3 signaling are vitamin D receptor-dependent. These data provide evidence that CYP24 limits 1,25(OH) 2 D 3 anti-proliferative signaling in cancer cells, and suggest that CTA091 may be beneficial in preserving 1,25(OH) 2 D 3 action in lung cancer.

The vitamin D(3) catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)(2)D(3)in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC-MS/MS assay that permits absolute quantitation of 1,25(OH)(2)D(3) in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vitro, and 1,25(OH)(2)D(3) rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)(2)D(3) reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20μg/kg 1,25(OH)(2)D(3)). When co-administered with the CYP24 selective inhibitor CTA091 (250μg/kg), 1,25(OH)(2)D(3) plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)(2)D(3) AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)(2)D(3) independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)(2)D(3) concentration versus time data confirmed that: 1,25(OH)(2)D(3) was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)(2)D(3).

We observed a 53% response rate in non-small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 microM) inhibited growth by: 17% +/- 7% in A549, 28% +/- 6% in 128-88T, 39% +/- 8% in Calu1 and 41% +/- 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 microM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128-88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 microM vorinostat, 83% +/- 10%; 5 microM carboplatin, 41% +/- 11%; carboplatin/vorinostat, 8% +/- 4%; 2 nM paclitaxel, 53% +/- 11%; paclitaxel/vorinostat, 46% +/- 21%. In A549 and 128-88T, vorinostat potentiated carboplatin induction of gamma-H2AX (a DNA damage marker) and increased alpha-tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in alpha-tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat-mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.

In non-small cell lung cancer (NSCLC) cells, 17beta-estradiol increases transcription, activates MAPK, and stimulates proliferation. We hypothesize that estrogen receptor beta (ERbeta) mediates these responses because it, but not ERalpha, is detected in our NSCLC cell lines. To test this, we determined the effects of the ERbeta-selective agonists genistein (GEN) and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) and the ERalpha-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) in 201T cells. The cells were transfected with either an ERalpha or an ERbeta expression vector and an estrogen response element (ERE)-tk-luciferase reporter construct. PPT increased luciferase activity in cells expressing ERalpha but not ERbeta. GEN and DPN selectively increased luciferase activity in ERbeta-transfected cells at concentrations < or =10 nM. Fulvestrant blocked the GEN- and DPN-mediated increases, indicating that transcription was ER-dependent. GEN but not PPT mediated a significant 1.5-fold increase in reporter activity upon transfection with ERE-tk-luciferase alone, demonstrating that endogenous ERbeta activates transcription. PPT and DPN increased MAPK phosphorylation (2.5-fold and 3.7-fold, respectively). However, only DPN stimulated 201T growth in vitro (p=0.008) and in vivo (p=0.05). We conclude that ERbeta mediates genomic and non-genomic responses to estrogen in 201T cells and that activation of both pathways may be necessary for increased proliferation of these cells.

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

Recent data support an important role for vitamin D in respiratory health. We tested the hypothesis that dietary vitamin D3 (VD3) intake modulates diaphragm (DIA) strength. Four-week-old female A/J mice ( n = 10/group) were randomized to receive diets containing 100 IU VD3/kg (low), 1,000 IU VD3/kg (reference), or 10,000 IU VD3/kg (pharmacologic). After 6 wk of dietary intervention, plasma 25-hydroxyvitamin D3 (25D3) levels, DIA and extensor digitorum longus (EDL) in vitro contractile properties, and fiber cross-sectional area (CSA) were measured. Myosin heavy chain (MHC) composition and Akt/Foxo3A growth signaling were studied in the DIA and tibialis anterior. Mice fed the low, reference, and pharmacologic diets had average 25D3 levels of 7, 21, and 59 ng/ml, respectively. Maximal DIA force, twitch force, and fiber CSA were reduced 26%, 28%, and 10% ( P < 0.01), respectively, in mice receiving the low-VD3 diet compared with the reference and pharmacologic diets. EDL force parameters were unaltered by diet. Effects of VD3 intake on DIA force were not observed in mice that began dietary intervention at 12 wk of age. VD3 intake did not alter the MHC composition of the DIA, indicating that decreases in force and CSA in young mice were not due to a switch in fiber type. Paradoxically, low VD3 intake was associated with activation of anabolic signaling in muscle (hyperphosphorylation of Akt and Foxo3A and decreased expression of autophagy marker LC3). These studies identify a potential role of dietary VD3 in regulating DIA development and insulin sensitivity.

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non–small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERα, ERβ, and ER coregulators in human lung fibroblasts and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERβ and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERα or the p160 coactivator SRC-1. ERβ and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist 17-β-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal growth factor, which served as a positive control for an alternative growth stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal growth factor also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

A novel class of pure antiestrogens, 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), lack estrogenic activity in a mouse uterotrophic assay and inhibit the growth of estrogen-sensitive MCF-7 breast cancer cells (Day et al., 1991). Here, reporter assays were used to evaluate the effects of the DTACs on estrogen receptor alpha (ERalpha)-mediated transcription from either classic estrogen-response elements (EREs) or nonclassic AP-1 elements. Among the DTACs tested, only the compounds with smaller aromatic substituents, BDRM72 and BDRM81, displayed weak agonist activity on EREs. Their activity was less than that observed for the ER partial agonist, 4-hydroxytamoxifen (ZOHT). In competition experiments, the DTACs blocked estradiol-stimulated transcription from an ERE in a dose-dependent manner and were more effective inhibitors than ZOHT. Each of the DTACs was significantly less active than ZOHT or the pure antiestrogen ICI 182,780 (faslodex) in stimulating transcription from nonclassic AP-1 elements in the presence of ERalpha. DTACs did not modulate either basal or TPA (12-O-tetradecanoylphorbol-13-acetate)-stimulated transcription from an AP-1 element in the absence of ERalpha, indicating that they are not nonspecific inhibitors of transcription and that ERalpha is the drug target. Glutathione S-transferase pull-down assays were used to examine whether DTACs alter the interaction between ERalpha and the p160 coactivator, GRIP1. BDRM35, which has the same dimethylaminomethoxy and phenolic moieties as ZOHT, reduced binding by more than 50%. Thus, disruption of p160 coactivator recruitment by ERalpha may represent one mechanism by which DTACs function as antiestrogens. BDRM35 also suppresses estradiol induction of endogenous target genes c-myc and cyclin D1 in MCF-7 breast cancer cells.

We have shown previously that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a normal prostate epithelial cell line is minimally affected by AITC even at concentrations that are highly cytotoxic to the prostate cancer cells. The present studies were designed to test the hypothesis that AITC administration may retard growth of human prostate cancer xenografts in vivo. Bolus i.p. injection of 10 micromol AITC, three times per week (Monday, Wednesday and Friday) beginning the day of tumor cell implantation, significantly inhibited the growth of PC-3 xenograft (P < 0.05 by two-way ANOVA). For example, 26 days after tumor cell implantation, the average tumor volume in control mice (1025 +/- 205 mm3) was approximately 1.7-fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the experiment revealed a statistically significant increase in number of apoptotic bodies with a concomitant decrease in cells undergoing mitosis in the tumors of AITC-treated mice compared with that of control mice. Western blot analysis indicated an approximately 70% reduction in the levels of anti-apoptotic protein Bcl-2 in the tumor lysate of AITC-treated mice compared with that of control mice. Moreover, the tumors from AITC-treated mice, but not control mice, exhibited cleavage of BID, which is known to promote apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2/M progression, including cyclin B1, cell division cycle (Cdc)25B and Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC administration inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and reducing mitotic activity.

Objectives The data reported are from a trial designed to determine, in patients with advanced cancer, the maximum tolerated dose and pharmacokinetics of calcitriol when administered with paclitaxel, an agent whose antitumor activity in in vitro and in vivo studies has been shown to be enhanced by calcitriol. An additional goal was to evaluate the relationship between calcitriol dose and hypercalcemia. Methods Calcitriol was given orally for 3 consecutive days each week, and paclitaxel (80 mg/m2) was given intravenously weekly. Thirty-six patients were treated in cohorts composed of 3 to 9 patients, at escalating dose levels of calcitriol. The starting dose of calcitriol was 4 μg for 3 consecutive days each week, and the maximum dose administered was 38 μg for 3 consecutive days each week. The preparation of calcitriol used in this trial was a commercially available caplet (0.5 μg per caplet). Serum calcitriol concentrations were measured by radioimmunoassay. Detailed assessments of calcitriol pharmacokinetics were performed in 26 patients. Results There was substantial interpatient variation in peak serum calcitriol concentrations (Cmax), time to reach Cmax, and area under the concentration versus time curve (AUC). Serum calcitriol AUC was not proportional to calcitriol dose (P = .0014). AUC for the 24-hour period after calcitriol administration [AUC (0–24)] at 38 μg was only 4 times that at 4 μg, instead of the 9.5-fold increase expected for a proportional relationship. Calcitriol plasma concentrations of 600 to 1440 pg/mL were achieved. No dose-limiting toxicity occurred in this trial. Conclusions Despite variability in absorption, very high doses of calcitriol can be safely administered with paclitaxel. The high calcitriol serum concentrations achieved in this study approach those that, both in vitro and in vivo, potentiate the cytotoxicity of taxanes and platinum analogs. Clinical Pharmacology & Therapeutics (2002) 72, 648–659; doi: 10.1067/mcp.2002.129305