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

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

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

mGluR1 transforms MCF10AT1 cells.

<p>MCF10AT1 cells, wild type or transduced with either <i>LacZ</i> or <i>GRM1</i>, were implanted into both flanks of athymic nude mice and allowed to grow for 8 weeks, after which MCF10AT1 lesions were harvested. <b>A. Representative wild type MCF10AT1 lesion</b> with both papillary enfolding (green arrow) and cribiform foci (red arrow). <b>B. </b><b><i>LacZ</i></b><b> control MCF10AT1 lesion.</b> The morphology indicates a hyperplastic lesion with both papillary (green arrow) and initial cribiforming present (red arrow). <b>C. mGluR1-overexpressing MCF10AT1 lesion.</b> The morphology indicates invasive cancer. These figures (<b>A</b> through <b>C</b>) are representative of 10 lesions analyzed for each group (magnification: 100×). <b>D. </b><b><i>GRM1</i></b><b> overexpression results in malignant transformation of MCF10AT1 cells.</b> Standard H&E sections of tumors were examined by a trained observer blinded to experimental group, and MCF10AT1 xenografts assessed for the presence and number of foci of carcinoma.</p

mGluR1 is active in a TNBC cell line.

<p>BT549 cells were stimulated in glutamate-free media containing GlutaMAX™ (Life Technologies, Grand Island, NY) with mGluR1 agonist, L-quisqualate (10 µM). Cells were harvested and the fold-increase in phosphorylated ERK1/2 was assayed by Western blot. Pretreatment with mGluR1 antagonist LY367385 for 30 minutes markedly reduced pERK1/2 induction by L-quisqualate (right side of gel). <b>Bottom gel:</b> Same blot stripped and re-probed with ERK antibody for normalization. Experiments were repeated two times with similar results.</p