Controversial relationship between the expression of the RB pathway components and RB protein phosphorylation in human breast cancer

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.     

RB1CC1 together with RB1 and p53 predicts long-term survival in Japanese breast cancer patients

RB1-inducible coiled-coil 1 (RB1CC1) plays a significant role in the enhancement of the retinoblastoma tumor suppressor (RB1) pathway and is involved in breast cancer development. However, RB1CC1's role in clinical progression of breast cancer has not yet been evaluated, so, as a first step, it is necessary to establish its usefulness as a tool to evaluate breast cancer patients. In this report, we have analyzed the correlation between abnormalities in the RB1CC1 pathway and long-term prognosis, because disease-specific death in later periods (>5 years) of the disease is a serious problem in breast cancer. Breast cancer tissues from a large cohort in Japan were evaluated by conventional immunohistochemical methods for the presence of the molecules involved in the RB1CC1 pathway, including RB1CC1, RB1, p53, and other well-known prognostic markers for breast cancer, such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The correlation between the immunohistochemical results and clinical outcomes of 323 breast cancer patients was analyzed using a Kaplan-Meier log-rank test and a multivariate Cox proportional hazards regression analysis. Absence of nuclear RB1CC1 expression was associated with the worst prognosis (Log-rank test, Chi-Square value = 17.462, p<0.0001). Dysfunction of either one of RB1CC1, RB1, or p53 was associated with the highest risk for cancer-specific death, especially related to survival lasting more than 5 years (multivariate Cox proportional hazard ratio = 3.951, 95% Confidence Interval =1.566-9.967, p = 0.0036). Our present data demonstrate that the combined evaluation of RB1CC1, RB1 and p53 by conventional immunohistochemical analysis provides an accurate prediction of the long-term prognoses of breast cancer patients, which can be carried out as a routine clinical examination.     

RB loss contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by multiple genetic events occurring in concert to drive pathogenic features of the disease. Here we interrogated the coordinate impact of p53, RB, and MYC in a genetic model of TNBC, in parallel with the analysis of clinical specimens. Primary mouse mammary epithelial cells (mMEC) with defined genetic features were used to delineate the combined action of RB and/or p53 in the genesis of TNBC. In this context, the deletion of either RB or p53 alone and in combination increased the proliferation of mMEC; however, the cells did not have the capacity to invade in matrigel. Gene expression profiling revealed that loss of each tumor suppressor has effects related to proliferation, but RB loss in particular leads to alterations in gene expression associated with the epithelial-to-mesenchymal transition. The overexpression of MYC in combination with p53 loss or combined RB/p53 loss drove rapid cell growth. While the effects of MYC overexpression had a dominant impact on gene expression, loss of RB further enhanced the deregulation of a gene expression signature associated with invasion. Specific RB loss lead to enhanced invasion in boyden chambers assays and gave rise to tumors with minimal epithelial characteristics relative to RB-proficient models. Therapeutic screening revealed that RB-deficient cells were particularly resistant to agents targeting PI3K and MEK pathway. Consistent with the aggressive behavior of the preclinical models of MYC overexpression and RB loss, human TNBC tumors that express high levels of MYC and are devoid of RB have a particularly poor outcome. Together these results underscore the potency of tumor suppressor pathways in specifying the biology of breast cancer. Further, they demonstrate that MYC overexpression in concert with RB can promote a particularly aggressive form of TNBC.     

RB pathway and therapeutic sensitivity: New insights in breast cancer and Tamoxifen therapy

Comment on: Lehn S, et al. Cell Cycle 2011; 10:956-962.     

RB1 and p53 at the crossroad of EMT and triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.     

Lack of RB protein correlates with increased sensitivity to UV-radiation-induced apoptosis in human breast cancer cells

The underlying causes for different apoptotic responses in neoplastic cells are still not fully understood. We demonstrate here that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (RB), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15-20-fold more sensitive to UV radiation than RB-positive cell lines, as measured by both apoptosis and clonogenic assays. In addition, a prostate cancer cell line that lacks functional RB, DU-145, was found to have a similar apoptotic response to low doses of UV radiation. Based on these data, we hypothesized that the lack of RB is responsible for the extreme sensitivity of these cells to UV-radiation-induced apoptosis. To further examine the role of RB in apoptosis, cells of RB-positive human breast cancer and normal cell lines were infected with the human papilloma virus type 16 (HPV-16) E7 and assessed for UV-radiation sensitivity. The HPV-16 E7 protein is known to decrease levels of free RB in cells. Infection of RB-positive human breast cancer or normal cells with E7 resulted in a 4-5-fold increase in sensitivity to UV radiation compared to controls. The above data suggest a role for the RB protein in protecting cells from undergoing apoptosis in response to UV radiation.     

A non-functional retinoblastoma tumor suppressor (RB) pathway in premenopausal breast cancer is associated with resistance to tamoxifen

The retinoblastoma tumor suppressor (RB) is important for retaining cell cycle control and loss of RB function is commonly observed in various malignancies. Experimental and animal studies have shown that RB knockdown in ER+ (estrogen receptor) cell lines and xenografts leads to resistance to tamoxifen, indicating that RB-inactivation could be linked to impaired response to specific cancer treatments. To address this issue, we utilized a unique randomized trial including 500 premenopausal breast cancer patients receiving either two years of adjuvant tamoxifen treatment or no treatment after surgery, and defined the tamoxifen response in RB-subgroups. Non-functional RB tumors were defined by lack of concordance between RB-phosphorylation and proliferation, in comparison to RB-functional tumors displaying comparable RB-phosphorylation and proliferation. In the ER+ tumors harboring a functional RB pathway (N=204), patients benefited from adjuvant tamoxifen with fewer breast cancer recurrences (HR=0.53, 95% CI 0.34-0.81, P=0.003). In the small subgroup of ER+ and RB non-functional tumors there was no benefit of tamoxifen (HR=2.28, 95% CI 0.51-10.3, P=0.28). In a multivariate analysis, the interaction between status of the RB pathway and treatment was significant (P=0.010), validating that despite being a small subgroup of ER+ breast cancer, RB functional status appears to be linked to response to tamoxifen treatment. These findings are in line with earlier experimental data altogether suggesting that analyses of RB status in breast cancer have the potential to be one among other future predictive factors that needs to be analyzed in order to successfully identify patients that will benefit from tamoxifen treatment.     

Impact of genetic variants in IL-2RA and IL-2RB on breast cancer risk in Chinese Han women

The expression of IL-2RA and IL-2RB was correlated with breast cancer (BC) progression. However, there is no literature investigating the association of IL-2RA and IL-2RB polymorphisms with BC predisposition among Chinese Han Women. Seven SNPs in IL-2RA and IL-2RB were genotyped by Agena MassARRAY platform among 553 BC patients and 550 healthy controls. Odds ratios (OR) and 95% confidence interval (CI) adjusted for age were calculated for the effect of IL-2RA and IL-2RB variants on BC susceptibility. IL-2RA rs12722498 was a protective factor for BC occurrence (OR?=?0.70, p?=?0.019), especially in subjects with age?≤?52 years (OR?=?0.55, p?=?0.004). IL-2RA rs12569923 (OR?=?9.07, p?=?0.033), IL-2RB rs2281089 (OR?=?0.67, p?=?0.043) and rs9607418 (OR?=?0.59, p?=?0.012) were related to the incidence of estrogen receptor positive (ER?+) BC. IL-2RB rs3218264 (OR?=?1.38, p?=?0.010) and rs9607418 (OR?=?0.56, p?=?0.009) were associated with the risk of developing progesterone receptor positive (PR?+) BC. Rs2281089 (OR?=?1.54, p?=?0.012) and rs1573673 (OR?=?0.72, p?=?0.035) were correlated to Ki-67 level. Moreover, IL-2RB rs2281089 (OR?=?0.72, p?=?0.022) showed a reduced risk of BC metastasis, and IL-2RA rs12722498 (OR?=?0.54, p?=?0.030) had a lower frequency in BC patients with tumor size?>?2 cm. Our study identified the potential effect of genetic variations in IL-2RA and IL-2RB on BC susceptibility and/or BC clinicopathologic indicators among Chinese Han Women.

     

Cytokines in breast cancer

In recent decades many advances have occurred in the understanding of the role of cytokines in breast cancer. New signalling pathways of interleukin (IL)-1 family, IL-6, IL-11, IL-18, interferons (IFNs) and interferon regulatory factors 1 (IRF-1) and 2 (IRF-2) have been found within tumour microenvironments and in metastatic sites. Some cytokines (IL-1, IL-6, IL-11, TGFbeta) stimulate while others (IL-12, IL-18, IFNs) inhibit breast cancer proliferation and/or invasion. Similarly, high circulating levels of some cytokines seem to be favourable (soluble IL-2R) while others are unfavourable (IL-1beta, IL-6, IL-8, IL-10, IL-18, gp130) prognostic indicators. So far IL-2, IFNalpha, IFNbeta and occasionally IFNgamma, IL-6, IL-12 have been the cytokines used for anti tumour treatment of advanced breast cancer either to induce or increase hormone sensitivity and/or to stimulate cellular immunity. Disappointing results occurred in most trials; however, two long-term pilot studies suggest that IL-2 and IFNbeta, when used appropriately can have a positive effect on clinical benefit and overall survival of patients with minimal residual disease after chemotherapy or with disseminated disease controlled by conventional endocrine therapy.     

Apoptosis in breast cancer

OBJECTIVE: To evaluate apoptotic rates on fine needle aspiration (FNA) samples of infiltrating duct carcinoma of the breast and to determine whether cytologic grading improved with consideration of the apoptotic rate in comparison with histologic grading. STUDY DESIGN: We studied 35 women who underwent mastectomy following an FNA diagnosis of infiltrating ductal carcinoma. Concordance between cytologic and histologic grades was calculated. Next, cytologic grades were considered with the apoptotic rates and compared with the histologic grades. RESULTS: An overall concordance of 82.9% was noted between the cytologic and histologic grading systems, with maximum concordance in grade 1 and minimum in grade 3 breast cancers. A highly significant difference in the apoptotic rates, as calculated on cytology, existed between the three histologic grades, indicating a significant increase in apoptosis with rising histologic grade. Applying multiple regression analysis, a significant improvement of cytologic grade with consideration of the apoptotic rate was observed. CONCLUSION: Employing histologic grade as the yardstick, cytology was less sensitive for the purpose of grading breast ductal carcinoma. However, by considering the apoptotic rates, the sensitivity of cytologic grading significantly rose in relation to histologic grade. Larger studies are required to determine whether apoptosis can be incorporated into the existing cytologic grading systems to increase their sensitivity.     

Primary and compensatory roles for RB family members at cell cycle gene promoters that are deacetylated and downregulated in doxorubicin-induced senescence of breast cancer cells

When treated with DNA-damaging chemotherapy agents, many cancer cells, in vivo and in vitro, undergo a terminal growth arrest and acquire a senescence-like phenotype. We investigated the molecular basis for this in breast cancer cells following a 2-hour treatment with 1 muM doxorubicin. Treated cells arrested in G1 and G2 phases of the cell cycle, with concomitant reductions in S-phase and G2-M regulatory genes. p53 and p21 protein levels increased within hours after treatment and were maintained for 5 to 6 days but were reduced 8 days posttreatment, though the cells remained growth arrested. Levels of p130 rose after drug treatment, and it was the primary RB family member recruited to the S-phase promoters cyclin A and PCNA and G2-M promoters cyclin B and cdc2, remaining present for the entire 8-day time period. In contrast, p107 protein and promoter occupancy levels declined sharply after drug treatment. RB was recruited to only the PCNA promoter. In MCF-7 cells with p130 knockdown, p107 compensated for p130 loss at all cell cycle gene promoters examined, allowing cells to retain the growth arrest phenotype. Cells with p130 and p107 knockdown similarly arrested, while cells with knockdown of all three family members failed to downregulate cyclin A and cyclin B. These results demonstrate a mechanistic role for p130 and compensatory roles for p107 and RB in the long-term senescence-like growth arrest response of breast cancer cells to DNA damage.     

Regulatory T cells increase in breast cancer and in stage IV breast cancer

Expression levels of VEGF and Her-2, levels of T-regulatory (Treg) cells, levels of CD3+ cells, and ratios of Th (CD4+ T cells)/Tr (Treg) cells were compared between stage I, II, III, and IV breast cancer patients (n?=?120) prior to chemotherapy and healthy women (n?=?30). Cells from peripheral blood were counted by flow cytometry, Her-2 and VEGF expression was detected by pathological examination, and Her-2 was detected by FISH. Breast cancer patients had more Treg cells and a lower ratio of Th/Tr cells than the healthy women. Stage IV breast cancer patients had more Treg cells and a lower ratio of Th/Tr cells than stage I, II, or III breast cancer patients. Patients positive for VEGF had a lower ratio of Th/Tr cells compared with patients negative for VEGF, and those positive for both VEGF and Her-2 also had a lower ratio of Th/Tr cells compared with patients not positive for both VEGF and Her-2. The decreased Th/Tr cells ratio indicates impaired immune function, suggesting that the stage IV breast cancer and the Her-2/VEGF-positive breast cancer patients have lower immune function.     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

Partial breast irradiation techniques in early breast cancer

Whole breast irradiation represents an integral part of combined breast-conserving treatment of early breast cancer. A new concept includes replacing traditionally fractionated whole breast postoperative radiotherapy by accelerated partial breast irradiation. The latter involves a variety of techniques and may be applied intraoperatively or shortly after the surgery. The intraoperative techniques include photon or electron external beam irradiation and interstitial high dose rate (HDR) brachytherapy, whereas the postoperative techniques comprise interstitial brachytherapy, be it HDR, pulse dose rate (PDR) or low dose rate (LDR), intracavitary brachytherapy and external beam radiotherapy using electrons, photons or protons. This article presents accelerated partial breast irradiation techniques, ongoing phase III trials evaluating their value and recommendations for clinical practice.     

Tumor-suppressive functions of 15-Lipoxygenase-2 and RB1CC1 in prostate cancer

15-Lipoxygenase-2 (15-LOX2) is a human-specific lipid-peroxidizing enzyme most prominently expressed in epithelial cells of normal human prostate but downregulated or completely lost in > 70% of prostate cancer (PCa) cases. Transgenic expression of 15-LOX2 in the mouse prostate surprisingly causes hyperplasia. Here we first provide evidence that 15-LOX2-induced prostatic hyperplasia does not progress to PCa even in p53^+/− or p53^−/− background. More important, by generating 15-LOX2; Hi-Myc double transgenic (dTg) mice, we show that 15-LOX2 expression inhibits Myc-induced PCa development, such that in the 3-month- and 6-month-old dTg mice, there is a significant reduction in prostate intraneoplasia (PIN) and PCa prevalent in age-matched Hi-Myc prostates. The dTg prostates show increased cell senescence and expression of several senescence-associated molecules, including p27, phosphorylated Rb, and Rb1cc1. We further show that in HPCa, 15-LOX2 and c-Myc manifest reciprocal protein expression patterns. Moreover, RB1CC1 accumulates in senescing normal human prostate (NHP) cells, and in both NHP and RWPE-1 cells, the 15-LOX2 metabolic products 15(S)-HPETE and 15(S)-HETE induce RB1CC1. We finally show that unlike 15-LOX2, RB1CC1 is not lost but rather frequently overexpressed in PCa samples. RB1CC1 knockdown in PC3 cells enhances clonal growth in vitro and tumor growth in vivo. Together, our present studies provide evidence for tumor-suppressive functions for both 15-LOX2 and RB1CC1.     

Relationship between the RB1 mRNA level and the expression of phosphorylated RB protein in human breast cancers: their relevance in cell proliferation activity and patient clinical outcome

The aim of the present study was to ascertain the relationship between the level of RB1 mRNA and the expression of phosphorylated RB protein and the relevance of these two parameters in cancer cell proliferation and clinical outcome in human breast cancer. Sixty-eight primary human breast cancers were considered. The amount of RB1 mRNA was evaluated by quantitative RT-PCR analysis. The level of RB phosphorylation was immunohistochemical defined by measuring the phosphorylated (pp) RB labelling index (LI). Cell proliferation rate was measured by calculating the Ki67 LI. No relation was found between the RB1 mRNA level and the ppRB LI (p=0.565). Both RB1 mRNA value and ppRB LI were related (in an inverse and direct manner, respectively) to Ki67 LI. RB1 mRNA expression was more strictly associated with KI67 LI (p=0.001) than the ppRB LI (p=0.013). Regarding the patient clinical outcome, the separately considered RB parameters did not reach the prognostic significance. However, patients with low RB1 mRNA quantity and patients with high ppRB LI, taken together, had a significantly shorter disease free and overall survival than the group comprehending patients with high RB1 mRNA value and low ppRB LI, and this despite the low number of patients considered. Our results demonstrated that the ppRB LI was independent of the RB1 mRNA level; that both RB parameters are related to the cell proliferation rate and, if collectively considered, have a high informative value on breast tumour prognosis.