Dave S.B. Hoon

FOXC1 Is a Potential Prognostic Biomarker with Functional Significance in Basal-like Breast Cancer

Gene expression signatures for a basal-like breast cancer (BLBC) subtype have been associated with poor clinical outcomes, but a molecular basis for this disease remains unclear. Here, we report overexpression of the transcription factor FOXC1 as a consistent feature of BLBC compared with other molecular subtypes of breast cancer. Elevated FOXC1 expression predicted poor overall survival in BLBC (P = 0.0001), independently of other clinicopathologic prognostic factors including lymph node status, along with a higher incidence of brain metastasis (P = 0.02) and a shorter brain metastasis-free survival in lymph node-negative patients (P < 0.0001). Ectopic overexpression of FOXC1 in breast cancer cells increased cell proliferation, migration, and invasion, whereas shRNA-mediated FOXC1 knockdown yielded opposite effects. Our findings identify FOXC1 as a theranostic biomarker that is specific for BLBC, offering not only a potential prognostic candidate but also a potential molecular therapeutic target in this breast cancer subtype.

BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition

Although targeting oncogenic mutations in the BRAF serine/threonine kinase with small molecule inhibitors can lead to significant clinical responses in melanoma, it fails to eradicate tumors in nearly all patients. Successful therapy will be aided by identification of intrinsic mechanisms that protect tumor cells from death. Here, we used a bioinformatics approach to identify drug-able, “driver” oncogenes restricted to tumor versus normal tissues. Applying this method to 88 short-term melanoma cell cultures, we show that the antiapoptotic BCL2 family member BCL2A1 is recurrently amplified in ∼30% of melanomas and is necessary for melanoma growth. BCL2A1 overexpression also promotes melanomagenesis of BRAF-immortalized melanocytes. We find that high-level expression of BCL2A1 is restricted to melanoma due to direct transcriptional control by the melanoma oncogene MITF. Although BRAF inhibitors lead to cell cycle arrest and modest apoptosis, we find that apoptosis is significantly enhanced by suppression of BCL2A1 in melanomas with BCL2A1 or MITF amplification. Moreover, we find that BCL2A1 expression is associated with poorer clinical responses to BRAF pathway inhibitors in melanoma patients. Cotreatment of melanomas with BRAF inhibitors and obatoclax, an inhibitor of BCL2A1 and other BCL2 family members, overcomes intrinsic resistance to BRAF inhibitors in BCL2A1-amplified cells in vitro and in vivo. These studies identify MITF-BCL2A1 as a lineage-specific oncogenic pathway in melanoma and underscore its role for improved response to BRAF-directed therapy.

Acetylated STAT3 is crucial for methylation of tumor-suppressor gene promoters and inhibition by resveratrol results in demethylation

The mechanisms underlying hypermethylation of tumor-suppressor gene promoters in cancer is not well understood. Here, we report that lysine acetylation of the oncogenic transcription factor STAT3 is elevated in tumors. We also show that genetically altering STAT3 at Lys685 reduces tumor growth, which is accompanied by demethylation and reactivation of several tumor-suppressor genes. Moreover, mutating STAT3 at Lys685 disrupts DNA methyltransferase 1–STAT3 interactions in cultured tumor cells and in tumors. These observations are confirmed by treatment with an acetylation inhibitor, resveratrol. Furthermore, reduction of acetylated STAT3 in triple-negative breast cancer cells leads to demethylation and activation of the estrogen receptor-α gene, sensitizing the tumor cells to antiestrogens. Our results also demonstrate a correlation between STAT3 acetylation and methylation of estrogen receptor-α in melanoma, which predicts melanoma progression. Taken together, these results suggest a role of STAT3 acetylation in regulating CpG island methylation, which may partially explain aberrant gene silencing in cancer. These findings also provide a rationale for targeting acetylated STAT3 for chemoprevention and cancer therapy.

Estrogen receptor and HER2/neu status affect epigenetic differences of tumor-related genes in primary breast tumors

IntroductionEstrogen receptor (ER)-positive breast cancers are considered prognostically more favorable than ER-negative tumors, whereas human epidermal growth factor receptor (HER)2/neu-positive breast cancers are associated with worse prognosis. The objective of the present study was to determine whether ER-positive and ER-negative status relates to epigenetic changes in breast cancer-related genes. To evaluate epigenetic differences in tumor-related genes relating to ER and HER2/neu status of primary tumors, we examined the promoter methylation status of the promoter region CpG islands of eight major breast tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RARβ2, and CDH1).MethodsPaired ER-positive (n = 65) and ER-negative (n = 65) primary breast tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted from paraffin-embedded tumor tissue after microdissection, and methylation-specific PCR and capillary-array electrophoresis analysis were performed.ResultsIn early stages of tumor progression (T1 and N0), RASSF1A and CCND2 were significantly (P < 0.05) more methylated in ER-positive than in ER-negative tumors. GSTP1 hypermethylation was more frequent in the lymph node metastasis positive group than in the negative group. Double negative (ER-negative, HER2/neu-negative) breast cancers had significantly lesser frequencies of RASSF1A, GSTP1, and APC methylation (P < 0.0001, P < 0.0001, and P = 0.0035, respectively). Both ER and HER2/neu status correlated independently with these epigenetic alterations.ConclusionWe demonstrated significant differences in tumor-related gene methylation patterns relevant to ER and HER2/neu status of breast tumors. This may be of significance in the assessment of targeted therapy resistance related to ER and HER2/neu status in breast cancer patients.

Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins

Background: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. Aims: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. Patients: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. Methods: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. Results: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; pu200a=u200a0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5–5.3), pu200a=u200a0.0009; and HR 2.8 (95% CI 1.4–5.5), pu200a=u200a0.004, respectively). Conclusions: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma.

Growth inhibition and modulation of cell markers of melanoma by S-allyl cysteine.

A sulfur-containing amino acid compound, S-allyl cysteine (SAC), derived from garlic extract inhibited proliferation of nine human and murine melanoma cell line in a dose-dependent manner (1.2-10 mM) assessed by a [3H]thymidine incorporation assay. Three control human lymphoblastoid cell lines were not inhibited by SAC concentrations < 5 mM. Four human melanoma cell lines in a soft-agar assay also showed dose-dependent inhibition of colony formation by SAC. Melanin content was increased up to 95% compared to the same untreated cell lines in these four human melanoma and two B16 murine melanoma sublines. Expression of cell surface gangliosides, cellular-differentiation and transformation markers, decreased after SAC treatment. Significant morphological changes including flattening and/or dendritic-like elongations were also observed. Thus SAC inhibited cellular growth and proliferation and modulated major cell differentiation markers of melanoma.

Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study

Long interspersed element 1 (LINE-1), a non-coding genomic repeat sequence, methylation status can influence tumor progression. In this study, the clinical significance of LINE-1 methylation status was assessed in primary breast cancer in young versus old breast cancer patients. LINE-1 methylation index (MI) was assessed by absolute quantitative assessment of methylated alleles (AQAMA) PCR assay. Initially, LINE-1 MI was assessed in a preliminary study of 235 tissues representing different stages of ductal breast cancer development. Next, an independent cohort of 379 primary ductal breast cancer patients (median follow-up 18.9xa0years) was studied. LINE-1 hypomethylation was shown to occur in DCIS and invasive breast cancer. In primary breast cancer it was associated with pathological tumor stage (pxa0=xa00.026), lymph node metastasis (pxa0=xa00.022), and higher age at diagnosis (>55, pxa0<xa00.001). In multivariate analysis, LINE-1 hypomethylation was associated with decreased OS (HR 2.19, 95xa0% CI 1.17–4.09, log-rank pxa0=xa00.014), DFS (HR 2.05, 95xa0% CI 1.14–3.67, log-rank pxa0=xa00.016) and increased DR (HR 2.83, 95xa0% CI 1.53–5.21, log-rank pxa0=xa00.001) in younger (≤55xa0years), but not older patients (>55 years). LINE-1 analysis of primary breast cancer demonstrated cancer-related age-dependent hypomethylation. In patients ≤55xa0years, LINE-1 hypomethylation portends a high-risk of DR.

Proteomic Profiling of Primary Breast Cancer Predicts Axillary Lymph Node Metastasis

To determine if protein expression in primary breast cancers can predict axillary lymph node (ALN) metastasis, we assessed differences in protein expression between primary breast cancers with and without ALN metastasis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Laser capture microdissection was performed on invasive breast cancer frozen sections from 65 patients undergoing resection with sentinel lymph node (SLN) or level I and II ALN dissection. Isolated proteins from these tumors were applied to immobilized metal affinity capture (IMAC-3) ProteinChip arrays and analyzed by SELDI-TOF-MS to generate unique protein profiles. Correlations between unique protein peaks and histologically confirmed ALN status and other known clinicopathologic factors were examined using ANOVA and multivariate logistic regression. Two metal-binding polypeptides at 4,871 and 8,596 Da were identified as significant risk factors for nodal metastasis (P = 0.034 and 0.015, respectively) in a multivariate analysis. Lymphovascular invasion (LVI) was the only clinicopathologic factor predictive of ALN metastasis (P = 0.0038). In a logistic regression model combining the 4,871 and 8,596 Da peaks with LVI, the area under the receiver operating characteristic curve was 0.87. Compared with patients with negative ALN, those with > or =2 positive ALN or non-SLN metastases were significantly more likely to have an increased peak at 4,871 Da (P = 0.016 and 0.0083, respectively). ProteinChip array analysis identified differential protein peaks in primary breast cancers that predict the presence and number of ALN metastases and non-SLN status.

Sequestosome 1 (SQSTM1) Mutations in Paget’s Disease of Bone from the United States

Paget’s disease of bone (PDB) is a localized bone disease characterized by excessive bone resorption due to overactive osteoclasts. Seven genetic loci (PDB1-PDB7) have been reported for late-onset PDB. PDB3 is the only locus where a gene, sequestosome 1 (SQSTM1), has been identified. Mutations in SQSTM1 have been associated with both sporadic and hereditary PDB in different populations. However, the SQSTM1 mutation frequency in PDB patients from a more heterogeneous population has never been reported. To investigate this, we determined the frequency of mutations in patients from the United States. Blood was collected from sporadic and hereditary PDB patients in the United States. DNA was isolated from whole blood or from serum. The SQSTM1 sequence was determined for exons and intron/exon junctions from whole blood and serum. A total of 112 (39 hereditary, 73 sporadic) samples were collected. Eight mutations were found in hereditary PDB patients, for a mutation frequency of 20.5% (95% confidence interval [CI] 10.8–35.5%) and did not differ significantly from mutation rates observed in studies in Canada, Great Britain, and The Netherlands. No mutations were found in sporadic patients, for a frequency of 0% (95% CI 0.0–5.0%), which was statistically significantly lower than the mutation rates previously observed in populations from Australia (Pxa0=xa00.009), Canada (Pxa0=xa00.008), Great Britain (Pxa0=xa00.02), and France (Pxa0=xa00.04) but not compared to rates from Belgium, The Netherlands, and Italy. Four out of five families with the P392L mutation carried it on the H2 haplotype. Mutations in SQSTM1 seem to contribute to the pathogenesis of PDB in hereditary, but not sporadic, patients in the United States.

A novel combination of suicide gene therapy and histone deacetylase inhibitor for treatment of malignant melanoma

One major problem associated with application of gene therapy to treatment of tumors is poor transgene expression. Although suicide gene therapy with the herpes simplex virus-thymidine kinase gene (HSV-tk) followed by administration of ganciclovir (GCV) was effective in the treatment of melanoma, it was still difficult to induce complete remission to cancer. A novel histone deacetylase inhibitor drug FR901229 was found to enhance transgene expression in tumor cells both in vitro and in vivo. Combination therapy with HSV-tklGCV and FR901228 by direct injection into tumor enhanced antimelanoma effects. The number of apoptotic cells in melanoma tumors was increased significantly (P<.05) after combined suicide gene therapy and FR901228. Six times injection of HSV-tk/GCV and FR901228 prolonged mice survival compared to that of HSV-tk/GCV injection alone (P=.021). In total, 56% (10 of 18) of the mice survived 120 days after combined suicide gene therapy and FR901228 treatment, and no new tumors appeared in the surviving mice. However, only 19% (3 of 16) of the mice survived when treated with suicide gene therapy alone. This novel strategy may be applicable as a therapeutic regimen for the treatment of aggressive types of cancers.