Survival analysis of circulating tumor cells in triple-negative breast cancer

Background. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008. [1] Triple-negative breast cancer (TNBC) is an aggressive phenotype comprising 10–20% of all breast cancers (BCs). [2-4] TNBCs show absence of estrogen, progesterone and HER2/neu receptors on the tumor cells. Because of the absence of these receptors, TNBCs are not candidates for targeted therapies. Circulating tumor cells (CTCs) are observed in blood of breast cancer patients even at early stages (Stage I & II) of the disease. Immunological and molecular analysis can be used to detect the presence of tumor cells in the blood (Circulating tumor cells; CTCs) of many breast cancer patients. These cells may explain relapses in early stage breast cancer patients even after adequate local control. CTC detection may be useful in identifying patients at risk for disease progression, and therapies targeting CTCs may improve outcome in patients harboring them. Methods . In this study we evaluated 80 patients with TNBC who are enrolled in a larger prospective study conducted at M D Anderson Cancer Center in order to determine whether the presence of circulating tumor cells is a significant prognostic factor in relapse free and overall survival . Patients with metastatic disease at the time of presentation were excluded from the study. CTCs were assessed using CellSearch System™ (Veridex, Raritan, NJ). CTCs were defined as nucleated cells lacking the presence of CD45 but expressing cytokeratins 8, 18 or 19. The distribution of patient and tumor characteristics was analyzed using chi square test and Fisher's exact test. Log rank test and Cox regression analysis was applied to establish the association of circulating tumor cells with relapse free and overall survival. Results. The median age of the study participants was 53years. The median duration of follow-up was 40 months. Eighty-eight percent (88%) of patients were newly diagnosed (without a previous history of breast cancer), and (60%) of patients were chemo naïve (had not received chemotherapy at the time of their blood draw for CTC analysis). Tumor characteristics such as stage (P=0.40), tumor size (P=69), sentinel nodal involvement (P=0.87), axillary lymph node involvement (P=0.13), adjuvant therapy (P=0.83), and high histological grade of tumor (P=0.26) did not predict the presence of CTCs. However, CTCs predicted worse relapse free survival (1 or more CTCs log rank P value = 0.04, at 2 or more CTCs P = 0.02 and at 3 or more CTCs P < 0.0001) and overall survival (at 1 or more CTCs log rank P value = 0.08, at 2 or more CTCs P = 0.01 and at 3 or more CTCs P = 0.0001. Conclusions. The number of circulating tumor cells predicted worse relapse free survival and overall survival in TNBC patients.^

Inflammatory Breast Cancer: The Immune Perspective

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Although rare and less than 2% of all breast cancer, IBC is responsible for up to 10% of all breast cancer deaths. Despite the name, very little is known about the role of inflammation or immune mediators in IBC. Therefore, we analyzed blood samples from IBC patients and non-IBC patients, as well as healthy donor controls to establish an IBC-specific profile of peripheral blood leukocyte phenotype and function of T cells and dendritic cells and serum inflammatory cytokines. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell-like features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. It is becoming increasingly evident that inflammatory factors can induce EMT. However, it is unknown if EMT-inducing soluble factors secreted by activated immune cells in the IBC microenvironment canπ account for the absence of EMT in studies of the tumor cells themselves. We hypothesized that soluble factors from immune cells are capable of inducing EMT in IBC. We tested the ability of immune conditioned media to induce EMT in IBC cells. We found that soluble factors from activated immune cells are able to induce the expression of EMT-related factors in IBC cells along with increased migration and invasion. Specifically, the pro-inflammatory cytokines TNF-α, IL-6 and TGF-β were able to induce EMT and blocking these factors in conditioned media abated the induction of EMT. Surprisingly, unique to IBC cells, this process was related to increased levels of E-cadherin expression and adhesion, reminiscent of the characteristic tightly packed tumor emboli seen in IBC samples. This data offers insight into the unique pathology of IBC by suggesting that tumor immune interactions in the tumor microenvironment contribute to the aggressive nature of IBC implying that immune induced inflammation can be a novel therapeutic target. Specifically, we showed that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. This data suggests that immune mediated inflammation may contribute to the highly aggressive nature of IBC and represents a potential therapeutic target that warrants further investigation.

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

The antiapoptotic effect of overexpression c -erBb2 in breast cancer

Overexpression of the receptor tyrosine kinase p185ErbB2 confers taxol resistance in breast cancers and activation of p34Cdc2 is required for taxol-induced apoptosis and cytotoxicity. Here, we investigated the underlying mechanisms and found that overexpression of p185 ErbB2 inhibits taxol-induced apoptosis through two branches to inhibit activation of p34Cdc2. ^ Overexpression of p185ErbB2 in MDA-MB-435 cells by transfection transcriptionally upregulated p21Cip1, which associates with p34Cdc2, inhibits taxol-mediated p34Cdc2 activation, delays cell entrance to G2/M phase, and thereby inhibits taxol-induced apoptosis. In p21Cip1 antisense-transfected MDA-MB-435 cells or in p21−/− MEF cells, p185ErbB2 was unable to inhibit taxol-induced apoptosis. Therefore, p21Cip1 participates in the regulation of a G2/M checkpoint that contributes to resistance to taxol-induced apoptosis in p185ErbB2-overexpressing breast cancer cells. ^ Direct phosphorylation on Tyrosine-15 of p34Cdc2 by p185 ErbB2 receptor tyrosine kinase inhibits p34Cdc2 activation. The wild-type p185ErbB2 but not the kinase-defective mutant, when overexpressed in breast cancer cells, can phosphorylate p34Cdc2 on tyrosine (Tyr)15, an inhibitory phosphorylation site of p34 Cdc2. The kinase domain of the ErbB2 receptor was sufficient for binding to p34Cdc2 and directly phosphorylating the recombinant Cdc2. Phosphospecific Cdc2-Tyr15 immunoblot analyses, immunocomplex kinase assays, and phospho-amino acid analyses revealed that p185ErbB2 specifically phosphorylates Cdc2 on Tyr15. Phosphorylation of Cdc2-Tyr15 by ErbB2 is modulated during cell cycle and corresponded with delayed cell entry into G2/M phase. The kinase-defective p185ErbB2, which incapable of phosphorylating Cdc2-Tyr15, failed to inhibit taxol-induced activation and apoptosis, whereas the wild-type and the constitutive-active p185ErbB2 did. Increased Cdc2-Tyr15 phosphorylation was found in Erb132-overexpressing tumors from breast cancer patients. Thus, direct phosphorylation of Cdc2-Tyr15 by p185 ErbB2 RTK in breast cancer cells inhibits taxol-induced p34 Cdc2 activation and apoptosis, thereby conferring taxol resistance. ^

Estradiol and insulin cross -talk in breast cancer

Approximately 33% of clinical breast carcinomas require estrogens to proliferate. Epidemiological data show that insulin resistance and diabetes mellitus is 2–3 times more prevalent in women with breast cancer than those with benign breast lesions, suggesting a clinical link between insulin and estradiol. Insulin and estradiol have a synergistic effect on the growth of MCF7 breast cancer cells, and long-term estradiol treatment upregulates the expression of the key insulin signaling protein IRS-1. The goal of this study was to further define the mechanism(s) of cross-talk between insulin and estradiol in regulating the growth of breast cancer. Using MCF7 cells, acute treatment with insulin or estradiol alone was found to stimulate two activities associated with growth: Erk MAP kinase and PI 3-kinase. However, combined acute treatment had an antagonistic effect on both activities. Acute estradiol treatment inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1 while increasing its serine phosphorylation; the serine phosphorylation was attenuated by the PI 3-kinase inhibitor wortmannin. The acute antagonism observed with combined estradiol and insulin are not consistent with the long-term synergistic effect on growth. In contrast, chronic estradiol treatment enhanced the insulin-sensitivity of breast cancer cells as measured by increases in total cellular insulin-stimulated tyrosine phosphorylation of IRS-1 and activation of PI 3-kinase. Estradiol stimulation of gene transcription was found to require PI 3-kinase activity but not MAP kinase activity. Insulin alone had no effect on ER transcriptional activity, but chronic treatment in combination with estradiol resulted in synergism of ER transcription. The synergistic effect of insulin and estradiol on MCF7 cell growth was also found to require PI 3-kinase but not MAP kinase activity. Therefore, chronic estradiol treatment increases insulin stimulation of PI 3-kinase, and PI 3-kinase is required for estradiol stimulation of gene transcription alone and in combined synergy with insulin. These data demonstrate that PI 3-kinase is the locus for the cross-talk between insulin and estradiol which results in enhanced breast cancer growth with long-term exposure to both hormones. This may have important clinical implications for women with high risk for breast cancer and/or diabetes mellitus. ^

Significance of tissue transglutaminase expression in multidrug resistant MCF -7 human breast cancer cells

Tissue transglutaminase (tTGase) is an enzyme that catalyzes the posttranslational modification of proteins via Ca2+-dependent cross-linking reactions. In this study, we extended our earlier observation that tTGase is highly expressed in MCF-7 human breast carcinoma cells selected for the multidrug resistance phenotype (MCF-7/DOX). To directly assess the involvement of tTGase in drug resistance, parental MCF-7 (MCF-7/WT) cells were transfected with cDNAs encoding either a catalytically active (wildtype) or inactive (mutant) tTGase protein. Expression of wildtype tTGase led to spontaneous apoptosis in MCF-7/WT cells, while the mutant tTGase was tolerated by the cells but did not confer resistance to doxorubicin. Analysis of calcium by a spectrofluorometric technique revealed that MCF-7/DOX cells exhibit a defective mechanism in intracellular calcium mobilization, which may play a role in preventing the in situ activation of tTGase and thus allowing the cells to grow despite expressing this enzyme. An elevation in intracellular calcium by treatment with the calcium ionophore A23187 induced rapid and substantial apoptosis in MCF-7/DOX cells as determined by morphological and biochemical criteria. Pretreatment of MCF-7/DOX cells with a tTGase-specific inhibitor (monodansylcadaverine) suppressed A12387-induced apoptosis, suggesting the possible involvement of tTGase-catalyzed protein cross-linking activity. A23187-induced apoptosis in MCF-7/DOX cells was further characterized by PARP cleavage and activation of downstream caspases (-3, -6, and -7). Another interesting aspect of tTGase/A23187-induced apoptosis in MCF-7/DOX cells was that these cells failed to show any prototypic changes associated with the mitochondrial (altered membrane potential, cytochrome c release, caspase-9 activation), receptor-induced (Bid cleavage), or endoplasmic reticulum-stressed (caspase-12 activation) apoptotic pathways. In summary, our data demonstrate that, despite being highly resistant to conventional chemotherapeutic drugs, MCF-7/DOX cells are highly sensitive to apoptosis induced by increased intracellular calcium. We conclude that tTGase does not play a direct role in doxorubicin resistance in MCF-7/DOX cells, but may play a role in enhancing the sensitivity of these cells to undergo apoptosis. ^