Johan Hartman

Clinically Used Breast Cancer Markers Such as Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 Are Unstable Throughout Tumor Progression

PURPOSE To investigate whether hormonal receptors and human epidermal growth factor receptor 2 (HER2) change throughout tumor progression, because this may alter patient management. PATIENTS AND METHODS The study cohort included female patients with breast cancer in the Stockholm health care region who relapsed from January 1, 1997, to December 31, 2007. Either biochemical or immunohistochemical (IHC)/immunocytochemical (ICC) methods were used to determine estrogen receptor (ER), progesterone receptor (PR), and HER2 status, which was then confirmed by fluorescent in situ hybridization for IHC/ICC 2+ and 3+ status. Results ER (459 patients), PR (430 patients), and HER2 (104 patients) from both primary tumor and relapse were assessed, revealing a change in 32.4% (McNemars test P < .001), 40.7% (P < .001), and 14.5% (P = .44) of patients, respectively. Assessment of ER (119 patients), PR (116 patients), and HER2 (32 patients) with multiple (from two to six) consecutive relapses showed an alteration in 33.6%, 32.0%, and 15.7% of patients, respectively. A statistically significant differential overall survival related to intraindividual ER and PR status in primary tumor and relapse (log-rank P < .001) was noted. In addition, women with ER-positive primary tumors that changed to ER-negative tumors had a significant 48% increased risk of death (hazard ratio, 1.48; 95% CI, 1.08 to 2.05) compared with women with stable ER-positive tumors. CONCLUSION Patients with breast cancer experience altered hormone receptor and HER2 status throughout tumor progression, possibly influenced by adjuvant therapies, which significantly influences survival. Hence, marker investigations at relapse may potentially improve patient management and survival.

Estrogen Receptor β Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERα and ERβ. ERα mediates the proliferative effect of estrogen in breast cancer cells, whereas ERβ seems to be antiproliferative. We engineered ERα-positive T47D breast cancer cells to express ERβ in a Tet-Off–regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERβ. The presence of ERβ resulted in a reduction in tumor growth. Comparison of the ERβ-expressing and non-ERβ–expressing tumors revealed that the expression of ERβ caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor β (PDGFβ). In cell culture, with the Tet-Off–regulated ERβ-expressing cells, expression of ERβ decreased expression of VEGF and PDGFβ mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFβ proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFβ promoter constructs revealed a repressive effect of ERβ at the promoter level of these genes. Taken together, these data show that introduction of ERβ into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)

Tumor repressive functions of estrogen receptor beta in SW480 colon cancer cells.

Estrogen receptor beta (ERbeta) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERbeta expression is reduced in colorectal cancer. Our hypothesis is that ERbeta inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERbeta in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERbeta expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERbeta resulted in inhibition of proliferation and G(1) phase cell cycle arrest and this effect was dependent on a functional DNA-binding region. c-Myc is overexpressed in an overwhelming majority of colorectal tumors. By Western blot and real-time PCR, we found c-Myc to be down-regulated in the ERbeta-expressing cells. Furthermore, the c-Myc target gene p21((Waf1/Cip1)) was induced and Cdc25A was reduced by ERbeta at the transcriptional level. The second cdk2-inhibitor, p27(Kip1), was induced by ERbeta, but this regulation occurred at the posttranscriptional level, probably through ERbeta-mediated repression of the F-box protein p45(Skp2). Expression of the ERbeta-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells +/- ERbeta transplanted into severe combined immunodeficient/beige mice; after three weeks of ERbeta-expression, a 70% reduction of tumor volume was seen. Our results show that ERbeta inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERbeta-mediated inhibition of cell-cycle pathways. Furthermore, this ERbeta-mediated cell cycle repression is dependent on functional ERE binding.

Estrogen receptor beta in breast cancer—Diagnostic and therapeutic implications

More than 10 years have passed since the discovery of the second estrogen receptor, estrogen receptor beta (ERbeta). It is now evident that ERalpha is not the only ER in breast cancer cells; in fact, ERbeta is expressed in the majority of breast cancers although at lower levels than in the normal breast. In addition, ERbeta is expressed in breast cancer infiltrating lymphocytes, fibroblasts and endothelial cells, all known to influence tumor growth. By overexpressing or knocking-out ERbeta in breast cancer cell lines, several researchers have investigated its function with respect to proliferation and tumor growth. It appears that ERbeta is anti-proliferative, in many ways antagonising the function of ERalpha. Furthermore, phytoestrogens have a binding-preference for ERbeta and several epidemiological studies indicate a breast cancer preventing effect of this class of compounds. Tamoxifen is one of the standard, adjuvant treatments for ERalpha positive breast cancer, classically thought to mediate its effect through ERalpha. However, in several recent studies, ERbeta has been described as a potential marker for tamoxifen response. In summary, experimental, epidemiological as well as diagnostic studies point towards ERbeta as an important factor in breast cancer, opening up the possibility for novel ERbeta-selective therapies in the treatment of breast cancer.

High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes

Background Breast cancer is a leading cause of death among women worldwide. Increasing evidence implies that human cytomegalovirus (HCMV) infection is associated with several malignancies. We aimed to examine whether HCMV is present in breast cancer and sentinel lymph node (SLN) metastases. Materials and Methods Formalin-fixed paraffin-embedded tissue specimens from breast cancer and paired sentinel lymph node (SLN) samples were obtained from patients with (n = 35) and without SLN metastasis (n = 38). HCMV immediate early (IE) and late (LA) proteins were detected using a sensitive immunohistochemistry (IHC) technique and HCMV DNA by real-time PCR. Results HCMV IE and LA proteins were abundantly expressed in 100% of breast cancer specimens. In SLN specimens, 94% of samples with metastases (n = 34) were positive for HCMV IE and LA proteins, mostly confined to neoplastic cells while some inflammatory cells were HCMV positive in 60% of lymph nodes without metastases (n = 35). The presence of HCMV DNA was confirmed in 12/12 (100%) of breast cancer and 10/11 (91%) SLN specimens from the metastatic group, but was not detected in 5/5 HCMV-negative, SLN-negative specimens. There was no statistically significant association between HCMV infection grades and progesterone receptor, estrogen receptor alpha and Elston grade status. Conclusions The role of HCMV in the pathogenesis of breast cancer is unclear. As HCMV proteins were mainly confined to neoplastic cells in primary breast cancer and SLN samples, our observations raise the question whether HCMV contributes to the tumorigenesis of breast cancer and its metastases.

Estrogen Receptors β1 and β2 Have Opposing Roles in Regulating Proliferation and Bone Metastasis Genes in the Prostate Cancer Cell Line PC3

The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.

HES-1 inhibits 17β-estradiol and heregulin-β1-mediated upregulation of E2F-1

We have previously shown that expression of the transcription factor HES-1 is required for the growth-inhibitory effect of all-trans retinoic acid on MCF-7 cells. In this study, we have used T47D cells with tetracyclin-regulated expression of wild-type or a dominant-negative form of HES-1. Expression of HES-1 in T47D cells inhibited G1/S-phase transition and activation of Cdk2 elicited by estrogen. Estrogen treatment of T47D cells caused increased expression of E2F-1, and this expression was inhibited by cotreatment with all-trans retinoic acid. We show that the effect is mediated through HES-1, which directly downregulates E2F-1 expression through a CACGAG-site within the E2F-1 promoter. Furthermore, proliferation caused by heregulin-β1 treatment of T47D cells was inhibited by all-trans retinoic acid and this effect was mediated by HES-1. Interestingly, heregulin-β1-mediated upregulation of E2F-1 expression was directly inhibited by HES-1 through the same CACGAG-site as seen with estrogen-stimulated induction. In addition, we found that two important downstream target genes of estrogen and heregulin-β1 that are regulated through E2F-1, cyclin E and NPAT, were both regulated in a similar fashion by all-trans retinoic acid, and these effects were antagonized by dominant-negative HES-1. These findings establish that HES-1 inhibits both estrogen- and heregulin-β1-stimulated growth of breast cancer cells, and further suggest that growth inhibition induced in these cells by all-trans retinoic acid occurs via HES-1-mediated downregulation of E2F-1 expression.

Current concepts and significance of estrogen receptor β in prostate cancer.

An increasing amount of evidence points at important roles for estrogen receptors in prostate carcinogenesis and progression. Of the two estrogen receptors, estrogen receptor β is the most prominent within the prostate gland. Although there is much yet to be known, the findings from the discovery of the receptor in 1996 until now point at a role of the receptor in maintaining differentiation and reducing cellular proliferation in the prostate. Moreover, estrogen receptor β is the main target for phytoestrogens, perhaps at least partially explaining the difference in incidence of prostate cancer in the Western world compared to Asia where the intake of soy-based, phytoestrogen-rich food is higher. The tumor suppressive capability of estrogen receptor β makes it a promising drug target for the treatment and prevention of prostate cancer. This review will focus on different aspects of estrogen receptor signaling and prostate cancer.

Digital image analysis outperforms manual biomarker assessment in breast cancer

In the spectrum of breast cancers, categorization according to the four gene expression-based subtypes ‘Luminal A,’ ‘Luminal B,’ ‘HER2-enriched,’ and ‘Basal-like’ is the method of choice for prognostic and predictive value. As gene expression assays are not yet universally available, routine immunohistochemical stains act as surrogate markers for these subtypes. Thus, congruence of surrogate markers and gene expression tests is of utmost importance. In this study, 3 cohorts of primary breast cancer specimens (total n=436) with up to 28 years of survival data were scored for Ki67, ER, PR, and HER2 status manually and by digital image analysis (DIA). The results were then compared for sensitivity and specificity for the Luminal B subtype, concordance to PAM50 assays in subtype classification and prognostic power. The DIA system used was the Visiopharm Integrator System. DIA outperformed manual scoring in terms of sensitivity and specificity for the Luminal B subtype, widely considered the most challenging distinction in surrogate subclassification, and produced slightly better concordance and Cohen’s κ agreement with PAM50 gene expression assays. Manual biomarker scores and DIA essentially matched each other for Cox regression hazard ratios for all-cause mortality. When the Nottingham combined histologic grade (Elston–Ellis) was used as a prognostic surrogate, stronger Spearman’s rank-order correlations were produced by DIA. Prognostic value of Ki67 scores in terms of likelihood ratio χ2 (LR χ2) was higher for DIA that also added significantly more prognostic information to the manual scores (LR−Δχ2). In conclusion, the system for DIA evaluated here was in most aspects a superior alternative to manual biomarker scoring. It also has the potential to reduce time consumption for pathologists, as many of the steps in the workflow are either automatic or feasible to manage without pathological expertise.

Novel Mechanism of Macrophage-Mediated Metastasis Revealed in a Zebrafish Model of Tumor Development

Cancer metastasis can occur at early stages of tumor development due to facilitative alterations in the tumor microenvironment. Although imaging techniques have considerably improved our understanding of metastasis, early events remain challenging to study due to the small numbers of malignant cells involved that are often undetectable. Using a novel zebrafish model to investigate this process, we discovered that tumor-associated macrophages (TAM) acted to facilitate metastasis by binding tumor cells and mediating their intravasation. Mechanistic investigations revealed that IL6 and TNFα promoted the ability of macrophages to mediate this step. M2 macrophages were particularly potent when induced by IL4, IL10, and TGFβ. In contrast, IFNγ-lipopolysaccharide-induced M1 macrophages lacked the capability to function in the same way in the model. Confirming these observations, we found that human TAM isolated from primary breast, lung, colorectal, and endometrial cancers exhibited a similar capability in invasion and metastasis. Taken together, our work shows how zebrafish can be used to study how host contributions can facilitate metastasis at its earliest stages, and they reveal a new macrophage-dependent mechanism of metastasis with possible prognostic implications.