Colleen A. Fordyce

Reduced telomere DNA content is correlated with genomic instability and metastasis in invasive human breast carcinoma.

Telomere shortening leads to genomic instability and has been correlated with poor outcome in several types of cancer. A recently described, robust titration assay was used to quantify telomere DNA content in frozen and paraffin-embedded specimens of 49 invasive human breast carcinomas, including tumors with normal or abnormal contents of genomic DNA, which produced regional, distant, or local disease. Telomere DNA contents ranged from 53% to 370% of the content in a reference DNA purified from normal placenta. Tumors were divided into three groups of approximately equal size based on increasing telomere DNA content. All of 16 tumors in the group with the least telomere DNA (Group I), were aneuploid compared to 9/17 tumors in the group with the most telomere DNA (Group III). The Chi-square test for trend indicated that tumors with the least telomere DNA were significantly more likely to be aneuploid than tumors with the most telomere DNA (p<0.002). Twelve of 14 tumors in Group I also produced metastatic disease compared to 8/15 tumors in Group III. The Fischer Exact Test indicated that tumors with the least telomere DNA were significantly more likely to be metastatic than tumors with the most telomere DNA (p<0.05). There was no association between telomere DNA content and patients’ age, tumors’ size, grade, stage, or fraction of cells in S-phase. The correlation of reduced telomere DNA content with aneuploidy and metastasis, both of which are associated with poor outcome in invasive breast carcinoma, implies that telomere DNA content also could have prognostic value.

Telomere DNA content and allelic imbalance demonstrate field cancerization in histologically normal tissue adjacent to breast tumors

Cancer arises from an accumulation of mutations that promote the selection of cells with progressively malignant phenotypes. Previous studies have shown that genomic instability, a hallmark of cancer cells, is a driving force in this process. In the present study, two markers of genomic instability, telomere DNA content and allelic imbalance, were examined in two independent cohorts of mammary carcinomas. Altered telomeres and unbalanced allelic loci were present in both tumors and surrounding histologically normal tissues at distances at least 1 cm from the visible tumor margins. Although the extent of these genetic changes decreases as a function of the distance from the visible tumor margin, unbalanced loci are conserved between the surrounding tissues and the tumors, implying cellular clonal evolution. Our results are in agreement with the concepts of “field cancerization” and “cancer field effect,” concepts that were previously introduced to describe areas within tissues consisting of histologically normal, yet genetically aberrant, cells that represent fertile grounds for tumorigenesis. The finding that genomic instability occurs in fields of histologically normal tissues surrounding the tumor is of clinical importance, as it has implications for the definition of appropriate tumor margins and the assessment of recurrence risk factors in the context of breast‐sparing surgery.

Telomere content correlates with stage and prognosis in breast cancer

SummaryPurposeTo evaluate the hypothesis that telomere DNA content (TC) in breast tumor tissue correlates with TNM staging and prognosis.Experimental designSlot blot assay was used to quantitate TC in 70 disease-free normal tissues from multiple organ sites, and two independent sets of breast tumors containing a total of 140 samples. Non-parametric Rank–Sums tests, logistic regression and Cox proportional hazards models were used to evaluate the relationships between TC and tumor size, nodal involvement, TNM stage, 5-year survival and disease-free interval.ResultsTC in 95% of normal tissues was 75–143% of that in the placental DNA standard, whereas only 50% of tumors had TC values in this range. TC was associated with tumor size (p=0.02), nodal involvement (p<0.0001), TNM stage (p=0.004), 5-year overall survival (p=0.0001) and 5-year disease-free survival (p=0.0004). A multivariable Cox model was developed using age at diagnosis, TNM stage and TC as independent predictors of breast cancer-free survival. Relative to the high TC group (>123% of standard), low TC (<101% of standard) conferred an adjusted relative hazard of 4.43 (95% CI 1.4–13.6, p=0.009). Receiver operating characteristic curves using thresholds defined by the TC distribution in normal tissues predicted 5-year breast cancer-free survival with 50% sensitivity and 95% specificity, and predicted death due to breast cancer with 75% sensitivity and 70% specificity.ConclusionsTC in breast cancer tissue is an independent predictor of clinical outcome and survival interval, and may discriminate by stage.

Inhibition of Ciliogenesis Promotes Hedgehog Signaling, Tumorigenesis, and Metastasis in Breast Cancer

Primary cilia are chemosensors that play a dual role to either activate or repress Hedgehog signaling, depending on presence or absence of ligand, respectively. While inhibition of ciliogenesis has been shown to be characteristic of breast cancers, the functional consequence is unknown. Here, for the first time, inhibition of ciliogenesis led to earlier tumor formation, faster tumor growth rate, higher grade tumor formation, and increased metastasis in the polyoma middle T (PyMT) mouse model of breast cancer. In in vitro model systems, inhibition of ciliogenesis resulted in increased expression of Hedgehog-target genes through a mechanism involving loss of the repressor form of the GLI transcription factor (GLIR) and activation of Hedgehog target gene expression through cross-talk with TGF-alpha (TGFA) signaling. Bioinformatics analysis revealed that increased Hedgehog signaling is frequently associated with increased TGFA; signaling in patients with triple-negative breast cancers (TNBC), a particularly aggressive breast cancer subtype. These results identify a previously unrecognized role for inhibition of ciliogenesis in breast cancer progression. This study identifies inhibition of ciliogenesis as an important event for activation of Hedgehog signaling and progression of breast cancer to a more aggressive, metastatic disease. Implications: These findings change the way we understand how cancer cells turn on a critical signaling pathways and a provide rationale for developing novel therapeutic approaches to target noncanonical Hedgehog signaling for the treatment of breast cancer. Mol Cancer Res; 15(10); 1421–30. ©2017 AACR.

F-actin reorganization by V-ATPase inhibition in prostate cancer

ABSTRACT The vacuolar ATPase (V-ATPase) proton pump sustains cellular pH homeostasis, and its inhibition triggers numerous stress responses. However, the cellular mechanisms involved remain largely elusive in cancer cells. We studied V-ATPase in the prostate cancer (PCa) cell line PC-3, which has characteristics of highly metastatic PCa. V-ATPase inhibitors impaired endo-lysosomal pH, vesicle trafficking, migration, and invasion. V-ATPase accrual in the Golgi and recycling endosomes suggests that traffic of internalized membrane vesicles back to the plasma membrane was particularly impaired. Directed movement provoked co-localization of V-ATPase containing vesicles with F-actin near the leading edge of migrating cells. V-ATPase inhibition prompted prominent F-actin cytoskeleton reorganization. Filopodial projections were reduced, which related to reduced migration velocity. F-actin formed novel cytoplasmic rings. F-actin rings increased with extended exposure to sublethal concentrations of V-ATPase inhibitors, from 24 to 48 h, as the amount of alkalinized endo-lysosomal vesicles increased. Studies with chloroquine indicated that F-actin rings formation was pH-dependent. We hypothesize that these novel F-actin rings assemble to overcome widespread traffic defects caused by V-ATPase inhibition, similar to F-actin rings on the surface of exocytic organelles. Summary: V-ATPase activates multiple stress responses. In prostate cancer, sub-lethal concentrations of V-ATPase inhibitors trigger widespread traffic defects. F-actin assembles into rings that mimic those seen during regulated exocytosis.

V-ATPase-dependent repression of androgen receptor in prostate cancer cells

Prostate Cancer (PCa) is the most commonly diagnosed cancer and the third leading cause of death for men in the United States. Suppression of androgen receptor (AR) expression is a desirable mechanism to manage PCa. Our studies showed that AR expression was reduced in LAPC4 and LNCaP PCa cell lines treated with nanomolar concentrations of the V-ATPase inhibitor concanamycin A (CCA). This treatment decreased PSA mRNA levels, indicative of reduced AR activity. V-ATPase-dependent repression of AR expression was linked to defective endo-lysosomal pH regulation and reduced AR expression at the transcriptional level. CCA treatment increased the protein level and nuclear localization of the alpha subunit of the transcription factor HIF-1 (HIF-1α) in PCa cells via decreased hydroxylation and degradation of HIF-1α. The addition of iron (III) citrate restored HIF-1α hydroxylation and decreased total HIF-1α levels in PCa cells treated with CCA. Moreover, iron treatment partially rescued CCA-mediated AR repression. Dimethyloxalylglycine (DMOG), which prevents HIF-1α degradation independently of V-ATPase, also decreased AR levels, supporting our hypothesis that HIF-1α serves as a downstream mediator in the V-ATPase-AR axis. We propose a new V-ATPase-dependent mechanism to inhibit androgen receptor expression in prostate cancer cells involving defective endosomal trafficking of iron and the inhibition of HIF-1 α-subunit turnover.

Vacuolar ATPase in Physiology and Pathology: Roles in Neurobiology, Infectious Disease, and Cancer

Vacuolar ATPase (V-ATPase) is an ATP-dependent proton pump present in all eukaryotic cells. V-ATPase is a critical regulator of intracellular pH across the endomembrane system and is essential for fundamental cellular functions including endocytosis and exocytosis, protein modification and maturation and loading of secretory vesicles. Here we describe the structure, regulation, and function of V-ATPase in pH regulation and the roles of V-ATPase in neurobiology, infectious disease, and cancer. V-ATPase is composed of two domains: a membrane-peripheral domain, V1, and a membrane-integral domain, Vo. When extracellular glucose concentrations drop the V1Vo complex disassembles to inhibit V-ATPase activity and prevent energy depletion; this ability allows yeast cells to quickly respond to alterations in energy state. Next, we present a body of growing new evidence that highlights the importance of V-ATPase in human health and disease. We discuss mechanisms by which V-ATPase participates in neurotransmission, neurodegeneration, and stroke-associated neuronal cell death. Then, we focus on the involvement of pH and V-ATPase in the pathogenesis of viruses, bacteria, and fungi and the processes necessary to ensure pathogen replication. In the last section, we capitalize upon a repertoire of studies in recent years that indicate that V-ATPase is a critical player in adaptation to cellular stress and that V-ATPase activity directly and indirectly contributes to many of the hallmarks of cancer.