Outgrowth of erlotinib-resistant subpopulations recapitulated in patient-derived lung tumor spheroids and organoids.

A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.

ACB-PCR Quantification of Low-Frequency Hotspot Cancer-Driver Mutations.

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive and quantitative approach for the selective amplification of a specific base substitution. Using the ACB-PCR technique, hotspot cancer-driver mutations (tumor-relevant mutations in oncogenes and tumor suppressor genes, which confer a selective growth advantage) are being developed as quantitative biomarkers of cancer risk. ACB-PCR employs a mutant-specific primer (with a 3'-penultimate mismatch relative to the mutant DNA sequence, but a double 3'-terminal mismatch relative to the wild-type DNA sequence) to selectively amplify rare mutant DNA molecules. A blocker primer having a non-extendable 3'-end and a 3'-penultimate mismatch relative to the wild-type DNA sequence, but a double 3'-terminal mismatch relative to the mutant DNA sequence is included in ACB-PCR to selectively repress amplification from abundant wild-type molecules. Consequently, ACB-PCR can quantify the level of a single base pair substitution mutation in a DNA population when present at a mutant:wild-type ratio of 1 × 10-5 or greater. Quantification of rare mutant alleles is achieved by parallel analysis of unknown samples and mutant fraction (MF) standards (defined mixtures of mutant and wild-type DNA sequences). The ability to quantify specific mutations with known association to cancer has several important applications in evaluating the carcinogenic potential of chemical exposures in rodent models. Further, the measurement of cancer-driver mutant subpopulations is important for precision cancer treatment (selecting the most appropriate targeted therapy and predicting the development of therapeutic resistance). This chapter provides a step-by-step description of the ACB-PCR methodology as it has been used to measure human PIK3CA codon 1047, CAT→CGT (H1047R) mutation.

Low-Frequency Mutational Heterogeneity of Invasive Ductal Carcinoma Subtypes: Information to Direct Precision Oncology.

Information regarding the role of low-frequency hotspot cancer-driver mutations (CDMs) in breast carcinogenesis and therapeutic response is limited. Using the sensitive and quantitative Allele-specific Competitor Blocker PCR (ACB-PCR) approach, mutant fractions (MFs) of six CDMs (PIK3CA H1047R and E545K, KRAS G12D and G12V, HRAS G12D, and BRAF V600E) were quantified in invasive ductal carcinomas (IDCs; including ~20 samples per subtype). Measurable levels (i.e., ≥ 1 × 10-5, the lowest ACB-PCR standard employed) of the PIK3CA H1047R, PIK3CA E545K, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E mutations were observed in 34/81 (42%), 29/81 (36%), 51/81 (63%), 9/81 (11%), 70/81 (86%), and 48/81 (59%) of IDCs, respectively. Correlation analysis using available clinicopathological information revealed that PIK3CA H1047R and BRAF V600E MFs correlate positively with maximum tumor dimension. Analysis of IDC subtypes revealed minor mutant subpopulations of critical genes in the MAP kinase pathway (KRAS, HRAS, and BRAF) were prevalent across IDC subtypes. Few triple-negative breast cancers (TNBCs) had appreciable levels of PIK3CA mutation, suggesting that individuals with TNBC may be less responsive to inhibitors of the PI3K/AKT/mTOR pathway. These results suggest that low-frequency hotspot CDMs contribute significantly to the intertumoral and intratumoral genetic heterogeneity of IDCs, which has the potential to impact precision oncology approaches.

Ovarian effects of prenatal exposure to benzo[a]pyrene: Roles of embryonic and maternal glutathione status.

Females deficient in the glutamate cysteine ligase modifier subunit (Gclm) of the rate-limiting enzyme in glutathione synthesis are more sensitive to ovarian follicle depletion and tumorigenesisby prenatal benzo[a]pyrene (BaP) exposure than Gclm+/+ mice. We investigated effects of prenatal exposure to BaP on reproductive development and ovarian mutations in Kras, a commonly mutated gene in epithelial ovarian tumors. Pregnantmice were dosed from gestational day 6.5 through 15.5 with 2mg/kg/day BaP or vehicle. Puberty onset occurred 5 days earlier in F1 daughters of all Gclm genotypes exposed to BaP compared to controls. Gclm+/- F1 daughters of Gclm+/- mothers and wildtype F1 daughters of wildtype mothers had similar depletion of ovarian follicles following prenatal exposure to BaP, suggesting that maternal Gclm genotype does not modify ovarian effects of prenatal BaP. We observed no BaP treatment or Gclm genotype related differences in ovarian Kras codon 12 mutations in F1 offspring.

Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas.

Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measureable levels of at least three of the five mutations. HRAS G12D was significantly increased in DCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10(-3) to 4.6 × 10(-2)) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047R mutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells.

Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation.

This study investigated whether Kras mutation is an early event in the development of lung tumors induced by inhalation of particulate vanadium pentoxide (VP) aerosols. A National Toxicology Program tumor bioassay of inhaled particulate VP aerosols established that VP-induced alveolar/bronchiolar carcinomas of the B6C3F1 mouse lung carried Kras mutations at a higher frequency than observed in spontaneous mouse lung tumors. Therefore, this study sought to: (1) characterize any Kras mutational response with respect to VP exposure concentration, and (2) investigate the possibility that amplification of preexisting Kras mutation is an early event in VP-induced mouse lung tumorigenesis. Male Big Blue B6C3F1 mice (6 mice/group) were exposed to aerosolized particulate VP by inhalation, 6h/day, 5 days/week for 4 or 8 weeks, using VP exposure concentrations of 0, 0.1, and 1 mg/m(3). The levels of two different Kras codon 12 mutations [GGT → GAT (G12D) and GGT → GTT (G12V)] were measured in lung DNAs by Allele-specific Competitive Blocker PCR (ACB-PCR). For both exposure concentrations (0.1 and 1.0mg/m(3)) and both time points (4 and 8 weeks), the mutant fractions observed in VP-exposed mice were not significantly different from the concurrent controls. Given that 8 weeks of inhalation of a tumorigenic concentration of particulate aerosols of VP did not result in a significant change in levels of lung Kras mutation, the data do not support either a direct genotoxic effect of VP on Kras or early amplification of preexisting mutation as being involved in the genesis of VP-induced mouse lung tumors under the exposure conditions used. Rather, the data suggest that accumulation of Kras mutation occurs later with chronic VP exposure and is likely not an early event in VP-induced mouse lung carcinogenesis.

Metabotropic glutamate receptor-1 contributes to progression in triple negative breast cancer.

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.

ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver.

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H-ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H-ras codon 61 CAA to AAA mutation increased in furan-treated mice. Allele-specific competitive blocker PCR (ACB-PCR) has been used previously to quantify early, carcinogen-induced increases in tumor-associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre-existing H-ras mutant cells in B6C3F1 mouse liver. H-ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H-ras codon 61 CTA or AAA mutant fraction (MF) greater than 10(-5) . Several furan-treated mice had H-ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically-significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H-ras mutant cells.

Metabotropic glutamate receptor-1: a potential therapeutic target for the treatment of breast cancer.

Metabotropic glutamate receptors are G-protein-coupled receptors normally expressed in the central nervous system where they mediate neuronal excitability, synaptic plasticity, and feedback inhibition of neurotransmitter release. However, recent data suggest that these receptors are also expressed and functional in some cancers, most notably melanoma. We detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in triple negative breast cancer cells and evaluated its role in regulating the pro-proliferative phenotype of these cells. mGluR1 inhibitors (Riluzole or BAY36-7620) inhibited the proliferation of triple negative breast cancer cells in a time- and dose-dependent manner and this inhibition correlated with increased apoptosis as demonstrated by increase in PARP cleavage products and Annexin V staining. mGluR1 knockdown using Lentiviral constructs expressing shRNA targeting GRM1 also inhibited proliferation compared to non-silencing controls. In addition, treatment of mice bearing MDA-MB-231 xenografts with Riluzole or BAY36-7620, by intraperitoneal injection, resulted in a significant reduction in tumor volume of up to 80%. Moreover, Riluzole was effective against triple negative breast cancer xenografts in mice at doses equivalent to those currently being used in humans for the treatment of amyotrophic lateral sclerosis. Our observations implicate mGluR1 and glutamate signaling as a promising new molecular target for the treatment of breast cancer. Even more promising, Riluzole, because it is an oral drug that can be administered with low toxicity, represents a promising approach in the treatment of triple negative breast cancer.

Antineoplastic effect of beta-elemene on prostate cancer cells and other types of solid tumour cells.

OBJECTIVES: beta-Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that beta-elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of beta-elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of beta-elemene on prostate cancer cell death was mediated through the induction of apoptosis. METHODS: The MTT assay was used to evaluate the ability of beta-elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA-based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl-2, caspases, cytochrome c and poly(ADP-ribose) polymerase (PARP) were analysed by Western blotting. KEY FINDINGS: Here, we showed that beta-elemene had an antiproliferative effect on androgen-insensitive prostate carcinoma DU145 and PC-3 cells. Treatment with beta-elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of beta-elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 microg/ml (230-465 microm). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by beta-elemene in a dose- and time-dependent manner. Moreover, beta-elemene exposure resulted in a decreased Bcl-2 protein level, increased cytochrome c release, and activated PARP and caspase-3, -7, -9, and -10 in prostate cancer cells. CONCLUSIONS: Overall, these findings suggest that beta-elemene exerts broad-spectrum antitumour activity against many types of solid carcinoma and supports a proposal of beta-elemene as a new potentially therapeutic drug for castration-resistant prostate cancer and other solid tumours.

Regulation of the expression and activity of the antiangiogenic homeobox gene GAX/MEOX2 by ZEB2 and microRNA-221.

Tumors secrete proangiogenic factors to induce the ingrowth of blood vessels from the stroma. These peptides bind to cell surface receptors on vascular endothelial cells (ECs), triggering signaling cascades that activate and repress batteries of downstream genes responsible for the angiogenic phenotype. To determine if microRNAs (miRNAs) affect regulation of the EC phenotype by GAX, a homeobox gene and negative transcriptional regulator of the angiogenic phenotype, we tested the effect of miR-221 on GAX expression. miR-221 strongly upregulated GAX, suggesting that miR-221 downregulates a repressor of GAX. We next expressed miR-221 in ECs and identified ZEB2, a modulator of the epithelial-mesenchymal transition, as being strongly downregulated by miR-221. Using miR-221 expression constructs and an inhibitor, we determined that ZEB2 is upregulated by serum and downregulates GAX, while the expression of miR-221 upregulates GAX and downregulates ZEB2. A mutant miR-221 fails to downregulate ZEB2 or upregulate GAX. Finally, using chromatin immunoprecipitation, we identified two ZEB2 binding sites that modulate the ability of ZEB2 to downregulate GAX promoter activity. We conclude that miR-221 upregulates GAX primarily through its ability to downregulate the expression of ZEB2. These observations suggest a strategy for inhibiting angiogenesis by either recapitulating miR-221 expression or inhibiting ZEB2 activation.

Evaluation and validation of housekeeping genes in response to ionizing radiation and chemical exposure for normalizing RNA expression in real-time PCR.

Gene expression changes are used with increasing frequency to assess the effects of exposure to environmental agents. Housekeeping (Hk) genes are essential in these analyses as internal controls for normalizing expression levels evaluated with Real-Time PCR (RT-PCR). Ideal Hk genes are constitutively expressed, do not respond to external stimuli and exhibit little or no sample-to-sample or run-to-run variation. Previous studies indicate that some commonly used Hk genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin have differential expression in various cell lines. Here we examine the expression of 11 Hk genes in four normal human lymphoblastoid cell lines and one T-cell leukemia (Jurkat) cell line following exposure to graded doses of ionizing radiation or to varying ratio concentrations of phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). PHA and PMA are known to have synergistic effects on the expression of some genes and have very different effects from those of radiation. There has been no systematic study performed to ascertain the best control genes for radiation and/or PHA/PMA exposures in lymphoblastoid cells. Using a two-step reverse-transcriptase RT-PCR protocol we show that following radiation doses ranging from 0 to 400 cGy, 18S rRNA, acidic ribosomal protein, beta-actin, cyclophilin, GAPDH, phosphoglycerokinase, beta-2 microglobulin (B2M), beta-glucuronidase, hypoxanthine phosphoribosyltransferase and transferrin receptor showed no significant variation in expression in normal lymphoblastoid cells. In contrast, only 18S rRNA levels were unchanged in Jurkat cells. After PHA/PMA treatment of the same normal cell lines, B2M showed no significant variation and 18S rRNA, GAPDH and transcription binding protein (TBP) were minimally responsive, whereas in Jurkat cells all these genes were unresponsive. While our results suggest that the utility of a particular Hk gene should be determined for each experimental condition, 18S rRNA and B2M appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells because they have the most constant levels of expression across cell lines following exposure to ionizing radiation as well as to PHA/PMA.