Exogenous normal mammary epithelial mitochondria suppress glycolytic metabolism and glucose uptake of human breast cancer cells.

We hypothesized that normal mitochondria inhibited cancer cell proliferation and increased drug sensitivity by the mechanism of suppression of cancer aerobic glycolysis. To demonstrate the mechanism, we used real-time PCR and glycolysis cell-based assay to measure gene expression of glycolytic enzymes and glucose transporters, and extracellular lactate production of human breast cancer cells. We found that isolated fluorescent probe-stained mitochondria of MCF-12A (human mammary epithelia) could enter into human breast cancer cell lines MCF-7, T47D, and MDA-MB-231, confirmed by fluorescent and confocal microscopy. Mitochondria from the untransformed human mammary epithelia increased drug sensitivity of MCF-7 cells to paclitaxel. Real-time PCR showed that exogenous normal mitochondria of MCF-12A suppressed gene expression of glycolytic enzymes, lactate dehydrogenase A, and glucose transporter 1 and 3 of MCF-7 and MDA-MB-231 cells. Glycolysis cell-based assay revealed that normal mitochondria significantly suppressed lactate production in culture media of MCF-7, T47D, and MDA-MB-231 cells. In conclusion, normal mitochondria suppress cancer proliferation and increase drug sensitivity by the mechanism of inhibition of cancer cell glycolysis and glucose uptake.

Progranulin (GP88) tumor tissue expression is associated with increased risk of recurrence in breast cancer patients diagnosed with estrogen receptor positive invasive ductal carcinoma.

INTRODUCTION: GP88 (progranulin) has been implicated in tumorigenesis and resistance to anti-estrogen therapies for estrogen receptor positive (ER+) breast cancer. Previous pathological studies showed that GP88 is expressed in invasive ductal carcinoma (IDC), but not in normal mammary epithelial tissue, benign lesions or lobular carcinoma. Based on these results, the present study examines GP88 prognostic significance in association with recurrence and death risks for ER+ IDC patients. METHODS: Two retrospective multi-site clinical studies examined GP88 expression by immunohistochemistry (IHC) analysis of paraffin-embedded breast tumor tissue sections from ER+ IDC patients (lymph node positive and negative, stage 1 to 3) in correlation with patients' survival outcomes. The training study established a GP88 cut-off value associated with decreased disease-free (DFS) and overall (OS) survivals. The validation study verified the GP88 cut-off value and compared GP88 prognostic information with other prognostic factors, particularly tumor size, grade, disease stage and lymph node status in multivariate analysis. RESULTS: GP88 expression is associated with a statistically significant increase in recurrence risk for ER+ IDC patients. The training study established that GP88 3+ score was associated with decreased DFS (P = 0.0004) and OS (P = 0.0036). The independent validation study verified that GP88 3+ score was associated with a 5.9-fold higher hazard of disease recurrence and a 2.5-fold higher mortality hazard compared to patients with tumor GP88 < 3+. GP88 remained an independent risk predictor after considering age, ethnicity, nodal status, tumor size, tumor grade, disease stage, progesterone receptor expression and treatments. CONCLUSIONS: The survival factor GP88 is a novel prognostic biomarker, predictive of recurrence risk and increased mortality for non-metastatic ER+ IDC patients. Of importance, our data show that GP88 continues to be a prognostic factor even after five years. These results also provide evidence that GP88 provides prognostic information independent of tumor and clinical characteristics and would support prospective study to examine whether GP88 expression could help stratify patients with ER+ tumors for adjuvant therapy.

Complete resolution of malignant ascites in stage IV breast cancer by peritoneal drainage and innovative chemoimmunotherapy: a case report.

Malignant ascites is a very serious and difficult to manage problem in the cancer patient. It is a sign of late stage disease and treatment is mainly palliative. Therefore the treatment should produce minimal discomfort and have few side effects. Numerous methods for managing the problem have been attempted, but few have afforded complete remission of the ascites with improved survival. We are reporting a case of complete remission of malignant ascites in a stage IV breast cancer patient. Her condition was managed by a peritoneal dialysis catheter and an aggressive intraperitoneal innovative chemoimmunotherapy protocol.

Determination of GP88 (progranulin) expression in breast tumor biopsies improves the risk predictive value of the Nottingham Prognostic Index.

BACKGROUND: The Nottingham Prognostic Index (NPI), which combines numerical values for nodal status, tumor size and histological grade, is used in the standard of care to provide predictive value information on post-surgery survival for patients with primary breast cancer. Attempts to improve the performance of the NPI algorithm have been carried out by testing the inclusion of other biomarker expression and morphological features such as vascular invasion. In the present study, we investigated whether expression of the autocrine growth and survival factor GP88 (progranulin), known to be overexpressed in breast cancer, would improve NPI's predictive value. METHODS: We examined by immunohistochemistry (IHC) the GP88 expression in 508 cases of estrogen receptor positive invasive ductal carcinoma with known clinical outcomes and for which NPI had been determined. GP88 IHC expression was scored by two board certified pathologists and classified into two score groups of GP88 <3+ (0, 1+, 2+) and GP88 = 3+. The correlation between GP88 scoring, NPI and disease-free (DFS) or overall survival (OS) outcomes was then examined by Kaplan-Meier analysis, Cox proportional Hazard (CPH) ratio and Pearson's X (2) test. RESULTS: Kaplan-Meier survival graphs of cases categorized by their NPI scores (<3.4, 3.4-5.4, >5.4) and GP88 expression showed that for patients within the same NPI subgroup, patients having tumors with a high GP88 expression (GP88 IHC score of 3+) had a worse DFS than patients with tumors that had a low GP88 expression (GP88 IHC score <3+). When adjusted for NPI, high GP88 score was significantly associated with recurrence with a hazard ratio of 3.30 (95 % CI 2.12 to 5.14). CONCLUSIONS: The data suggest that the determination of GP88 tumor expression at time of diagnosis for early stage breast cancer patients can provide additional survival information to that provided by NPI alone and thus may be useful for risk management of patients diagnosed with breast cancer.

Antisense ferritin oligonucleotides inhibit growth and induce apoptosis in human breast carcinoma cells.

BACKGROUND: Ferritin is the major iron-storage protein which sequesters and detoxifies excess iron that is taken up by cells but is not utilized in normal metabolic processes. Human ferritin consists of various combinations of heavy (FerH, Mr 21,000) and light (FerL, Mr 19,000) chains and excess iron leads to an increase in the synthesis of both heavy and light chains. MATERIALS AND METHODS: In this study four pairs of antisense oligodeoxynucleotides (ODNs) were synthesized: FerH-A1 and FerL-A1 were complementary to the 24-base pair sequence overlapping the starting codons of the FerH and FerL genes, respectively, but the sequences of FerH-A2 and FerL-A2 only covered the coding sequences of the ferritin genes. The corresponding sense chain sequences (FerH-S1, FerH-S2, FerL-S1 and FerL-S2) were used as controls. RESULTS: Treatment with FerH-S1, FerH-A1, FerH-S2, FerH-A2, FerL-S1, FerL-A1, FerL-S2 and FerL-A2 at 40 microM, 25 microM, 30 microM, 17 microM, 45 microM, 18 microM, 40 microM and 26 microM, respectively, for 72 hours resulted in 50% inhibition of DNA synthesis (IC50) in MCF-7 breast carcinoma cells, as measured by [3H]-thymidine incorporation. FerH chain mRNA, FerL chain mRNA and total ferritin protein levels were significantly decreased by the IC50 concentrations of each of the antisense ODNs but were not inhibited by IC50 concentrations of sense ODNs, as measured by quantitative RT-PCR and microparticle enzyme immunoassay. However, antisense ferritin ODNs had no effect on the total iron concentration in MCF-7 cells. Incubation with IC50 concentrations of antisense ferritin ODNs caused reduction in cell volume, condensation of nuclear structures and lower levels of Bcl-2 mRNA and protein compared to control cells, but Bax mRNA and protein levels remained unchanged. CONCLUSION: This study demonstrates that antisense ODNs to ferritin genes are about two-fold more cytotoxic than sense ODNs, and that antisense ODNs are specific inhibitors of ferritin gene expression at both the transcriptional and the translational levels. Further, the antisense ferritin ODNs promote programmed cell death with low ratios of Bcl-2 to Bax mRNA and protein expression providing evidence that antisense ferritin ODNs specifically inhibit MCF-7 breast carcinoma cell growth through increased apoptosis. Finally, since the IC50 concentrations of FerH-A1 and FerH-A2, and FerL-A1 and FerL-A2 are very similar for inhibition of DNA synthesis and gene expression in human breast carcinoma MCF-7 cells, it does not seem necessary for the antisense ODNs to overlap the starting codons of ferritin gene to maximize inhibition.

Induction of apoptosis by iron depletion in the human breast cancer MCF-7 cell line and the 13762NF rat mammary adenocarcinoma in vivo.

It is known that the interruption of normal iron metabolism with chelators of iron, toxic metals, toxic metals bound to transferrin, or anti-transferrin receptor antibodies leads to significant inhibition of tumor cell growth in cell culture systems and animal models. In the present study, we found that iron depletion was produced by the iron chelator deferoxamine mesylate, the free toxic metals gallium or indium, and the toxic metals gallium or indium bound to transferrin in the MCF-7 human breast cancer cell line, and this induced the condensation and fragmentation of chromatin, and the formation of DNA fragments characteristic of apoptosis. The induction of apoptosis was quantitated with acridine orange and ethidium bromide staining of apoptotic cells, separation of fragmented DNA from radiolabeled cells, and in situ terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assays. The apoptosis, caused by deferoxamine mesylate, and gallium or indium bound to transferrin in the MCF-7 cells, can be completely inhibited by excess ferric chloride or equimolar iron-loaded transferrin. Gallium-transferrin and indium-transferrin complexes induced more apoptosis than their respective salts in the MCF-7 cells. Deferoxamine mesylate induced a small increase in the endogenous expression of both the bcl-2 and bax genes in the MCF-7 cells and this can be prevented by ferric chloride. In the 13762NF rat mammary adenocarcinoma model, in situ TUNEL assays showed that the iron-deficiency following a low iron diet or intravenous injection of deferoxamine mesylate produced 5.32 +/- 3.90% and 6.46 +/- 3.58% of apoptotic cells, respectively, compared to 2.01 +/- 1.20% of apoptotic cells in the control rats maintained on a normal diet (p < 0.05 and p < 0.01, respectively, Student's t-test). This is the first report of iron depletion caused by a low iron diet or deferoxamine mesylate treatment inducing apoptosis in rats bearing the 13762NF marnmary adenocarcinoma.

Radiofrequency ablation of a stereotactically localized nonpalpable breast carcinoma.

A nonpalpable breast lesion was detected in a 71-year-old woman who had returned for her annual mammogram. Stereotactic core needle biopsy revealed an infiltrating ductal carcinoma. The patient agreed to stereotactic localization and radiofrequency ablation of the lesion followed after 4 weeks by open surgical biopsy. The breast lesion was localized and the radiofrequency ablation performed under local anesthesia in the outpatient/office setting. The lesion was ablated for a total of 20 minutes at a sustained mean temperature of 75 degrees C. After a 30-second cooldown the peripheral temperature of the four peripheral thermocouples ranged from 58 degrees C to 70 degrees C. A surgical clip was placed at the site of the ablated lesion. The postprocedure course was uneventful and the patient proceeded to open biopsy 4 weeks later. The open biopsy specimen, a left segmental mastectomy, underwent specimen radiography, which confirmed the surgical clip in the center of the lesion. There was extensive central necrosis and hemorrhage surrounded by fat necrosis. There was no definite viable residual tumor and the margins were clear. This is the first case in a clinical protocol designed to determine the efficacy of stereotactic localization and radiofrequency ablation of nonpalpable breast lesions. Additional ablations will be required to define the procedure but the results from this initial patient suggest that this is a promising minimally invasive curative approach for nonpalpable breast lesions.

Adjuvant breast cancer vaccine improves disease specific survival of breast cancer patients with depressed lymphocyte immunity.

PURPOSE: Beginning in 1995 breast cancer patients were vaccinated in the adjuvant setting with an autologous, allogeneic whole cell vaccine to evaluate the effect on host lymphocyte immunity and disease specific survival. METHODS: The breast cancer patients had host lymphocyte immunity against tumor associated antigens evaluated by a Lymphocyte Blastogenesis Assay (LBA) before vaccination. Thirty-seven patients with depressed immunity were vaccinated in the adjuvant setting. Patients were given six intradermal injections (three weekly followed by three monthly). Ten weeks after the last injection the LBA was repeated. RESULTS: Some patients experienced slight pain and swelling at the injection site with slight chills and fever, but there were no severe toxicities. The vaccinated patients had a mean follow-up of 12.7 years with mean follow-up of 8.9 and 9.2 years for the patients with normal and depressed immunity, respectively, in the historic control. The 10 year survival was 95% (20 of 21 patients) in the normal immunity historic control, 59% (33 of 56 patients) in the depressed immunity historic control and 89% (33 of 37 patients) in the patients with depressed immunity that were vaccinated in the present clinical trial. The disease specific survival of the vaccinated patients with depressed immunity in this trial is significantly greater than that of the historic controls of unvaccinated patients with depressed immunity to their tumor associated antigens. CONCLUSION: This study confirms the importance of maintaining good host lymphocyte immunity after completion of standard therapy and validates the value of cancer immunotherapy in the adjuvant setting.

Down-regulation of expression of interleukin-6 and its receptor results in growth inhibition of MCF-7 breast cancer cells.

Interleukin-6 (IL-6) plays an important role in the neoplastic process through its action on cancer cell adhesion, motility, proliferation, tumor-specific antigen expression, and thrombopoiesis. IL-6 exerts its activity by binding to a high affinity receptor complex consisting of two membrane glycoproteins: the 80 kDa IL-6 a-receptor subunit (IL-6R) and the 130 kDa signal-transducing protein (GP130). In the present study, MCF-7 breast cancer cells were cultured with human IL-6 and IL-6 soluble receptor (sIL-6R). MCF-7 cells were also treated with either antibodies specific to human IL-6 and IL-6R, or synthetic antisense oligodeoxynucleotides (ODNs) targeted to IL-6 and IL-6R genes. Cell growth was measured, and it was found that human IL-6 and sIL-6R did not significantly increase the proliferation of MCF-7 cells. When IL-6 produced by the MCF-7 cells was bound by rabbit anti-human IL-6 antibody, there was a significant dose-dependent inhibition of cell proliferation. IL-6 and IL-6R antisense ODNs caused a marked and specific decrease in IL-6 and IL-6R mRNA and proteins, respectively. Both IL-6 and IL-6R antisense ODNs significantly inhibited the proliferation of MCF-7 cells, but the inhibitory effect of IL-6R antisense ODN was greater than that of IL-6 antisense ODN (IC50: IL-6R: 1 µM; IL-6: 5 µM, 72-hour incubation). Addition of exogenous IL-6 partially reversed the growth inhibition caused by IL-6 antisense ODN but not the growth inhibition caused by IL-6R antisense ODN. In conclusion, IL-6 plays an important role in maintaining the growth of MCF-7 breast cancer cells. These results suggest careful modulation of IL-6 and IL-6R expression of cells as a potential approach for breast cancer therapy.

Human leukocyte antigen G expression in breast cancer: role in immunosuppression.

Human leukocyte antigen G (HLA-G) is an immunotolerant nonclassical major histocompatibility complex Class Ib molecule. It is expressed by trophoblastic placental cells during pregnancy to protect the fetus from maternal alloreactivity. HLA-G is overexpressed in tumors and involved in cancer immune evasion. Reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were used to examine HLA-G expression in normal mammary and breast cancer cell lines and normal and human breast cancer tissues. Reverse transcription-polymerase chain reaction confirmed that normal epithelial MCF-12A cells had no HLA-G mRNA expression, whereas cancer cell lines MCF-7, T47D, and MDA-MB-231 and NCI/Adr-Res had various levels of HLA-G mRNA expression. Twelve (12) normal and 38 breast cancer tissues were examined by IHC. Fifty-eight (58) percent (22/38) of cancers had medium to strong staining to HLA-G, whereas only 8% (1/12) of normal breast tissues had medium to strong staining, and the difference was significant (p < 0.05). HLA-G staining was found in the membranes and cytoplasm of cancer cells. In conclusion, breast cancer cells overexpress HLA-G mRNA and protein, and this probably contributes to immune evasion.

Manipulation of iron transporter genes results in the suppression of human and mouse mammary adenocarcinomas.

Since malignant cells often have a high demand for iron, we hypothesize that breast cancer cells may alter the expression of iron transporter genes including iron importers [transferrin receptor (TFRC) and solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (SLC11A2)] and the iron exporter SLC40A1 (ferroportin), and additionally that the growth of breast cancer can be inhibited by manipulating iron transporter gene expression. To test our hypothesis, reverse transcription polymerase chain reaction (RT-PCR) was used to determine mRNA expression of iron transporter genes in normal human mammary epithelial MCF-12A cells and human breast cancer MCF-7 cells. Antisense oligonucleotides were employed to suppress the expression of TFRC gene in the 4T1 mammary adenocarcinoma in both cell culture and a mouse tumor model. We found the following: i) the MCF-7 cells have higher expression of TFRC and SLC11A2 compared with MCF-12A epithelia; ii) SLC40A1 was only expressed in MCF-12A epithelia but not in MCF-7 cells; iii) iron increased mRNA levels of the SLC11A2 gene in both MCF-12A and MCF-7 cells; iv) TFRC antisense oligonucleotides reduced TFRC mRNA levels and intracellular total iron, and inhibited the proliferation of the 4T1 cells in cell culture; v) TFRC antisense oligonucleotide inhibited tumor growth and lung metastases in the 4T1 mammary adenocarcinoma mouse model. In conclusion, breast cancer cells up-regulate the expression of iron importer genes and down-regulate the expression of iron exporter SLC40A1 to satisfy their increased demand for iron. Suppression of transferrin receptor by antisense results in inhibition of tumor growth and lung metastasis in the 4T1 mammary adenocarcinoma mouse model.

Stereotactic radiofrequency ablation: a minimally invasive technique for nonpalpable breast cancer in postmenopausal patients.

BACKGROUND: The purpose of this study was to determine the feasibility of radiofrequency ablation (RFA) of nonpalpable breast cancer in postmenopausal women, and report on long-term follow-up with clinical examination and mammography. METHODS: Since November 2000, we have performed RFA on stereotactically localized nonpalpable breast cancers (only mammographic densities) in women older than 65 years with other serious health problems. RESULTS: The first patient had the procedure done in the office with sedation and local anesthesia. The radiofrequency probe was inserted by stereotactic localization, and the RFA proceeded for 20 min at 75 degrees C. Two weeks later, the lesion was not seen mammographically, but by palpation there was induration at the ablation site. Six weeks later, open excision of the area confirmed a prominent ablation site with no remaining viable tumor cells. The second patient had the same procedure, and has been followed without open biopsy. The third patient had DCIS and the probe arrays were not able to penetrate the lesion. The fourth and fifth patients had light sedation with an intercostal nerve block to eliminate discomfort and this approach was found to be a very effective office procedure. The last two patients' ablation sites were injected with depo-medrol and they were placed on anti-inflammatory therapy to decrease the palpable induration caused by fat necrosis. CONCLUSION: We found RFA feasible for definitive therapy for nonpalpable breast cancer. If our results are confirmed by larger clinical trials, RFA would eliminate open surgery and decrease the morbidity associated with lumpectomy and radiation.

Gene targets of antisense therapies in breast cancer.

Advances in molecular and cell biology have led to further understanding of the mechanisms of malignant growth and metastasis in human breast cancer cells. Initiation and progression of breast cancer results from mutations and the abnormal expression of many genes that control cellular proliferation, differentiation, invasion, metastasis and sensitivity to therapy (chemotherapy and radiation therapy). Inhibition of host immunity also plays a role in breast cancer progression. Many genes have been selected as targets for antisense therapy, including HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR, P12, MDM2, BRCA, Bcl-2, ER, VEGF, MDR, ferritin, transferrin receptor, IRE, C-fos, HSP27, C-myc, C-raf and metallothionein genes. The strategy behind antisense therapy is the development of specific therapeutic agents that aim to correct the mutations and abnormal expression of cellular genes in breast tumour cells by decreasing gene expression, inducing degradation of target mRNA and causing premature termination of transcription. Many in vitro and in vivo studies have investigated the therapeutic efficacy of oligonucleotides and antisense RNAs. These studies have demonstrated specific inhibition of tumour cell growth by antisense therapy and have shown synergistic inhibitory effects between antisense oligonucleotides or antisense RNA and conventional chemotherapeutic drugs used in the treatment of breast cancer. Antisense oligonucleotides have been modified to improve their ability to penetrate cells, bind to gene sequences and downregulate target gene function. Many delivery systems for antisense RNA and antisense oligonucleotides have been developed, including virus vectors (retrovirus, adenovirus and adeno-associate virus) and liposomes, to carry the antisense RNA or oligonucleotides through the cell membrane into the cytoplasm and nucleus of the tumour cells. However, in order to determine their feasibility antisense therapies need to be further investigated to determine their antitumour activity, pharmacokinetics and toxicity in breast cancer patients.