Peritumoral FOXP3+ regulatory T cell is sensitive to chemotherapy while intratumoral FOXP3+ regulatory T cell is prognostic predictor of breast cancer patients

It has been reported that the prognostic significances of tumor-infiltrating FOXP3(+) regulatory T cells (Tregs) in breast carcinoma depend on their relative density and tissue locations. We here assessed the changes of Tregs before and after neoadjuvant chemotherapy (NC) and their relationships with tumor response and patient survival. Intratumoral and peritumoral infiltration of FOXP3(+) Tregs were evaluated by immunohistochemistry in 132 cases of invasive breast carcinomas before and after NC. After NC, the density of infiltrated Tregs within tumor bed remained stable, whereas it decreased significantly (P?=?0.015) in the tissue surrounding tumor. The changes were significant in those tumors that usually response to NC, including the HER2-enriched and basal-like subtypes (P?=?0.035; P?=?0.004). Univariate and multivariate analyses identified the decreased peritumoral Tregs were an independent predictor for pathologic complete response (pCR), while the intratumoral Tregs after chemotherapy was proved to be associated with overall survival and progression-free survival of the patients. The findings of the study indicated that peritumoral Treg was sensitive to chemotherapy and associated with pCR, while intratumoral Treg was an independent prognostic predictor of breast cancer patients.     

Functional heterogeneity of circulating T regulatory cell subsets in breast cancer patients

Background

Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women.

Methods

The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4⁺CD25⁺FoxP3⁺CD45RA⁺ N nTregs, CD4⁺CD25⁺FoxP3⁺CD45RA^? EM nTregs, and CD4⁺IL-4^?IL-10⁺ Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry.

Results

Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25⁺ and FoxP3⁺ Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25⁺ Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I–III).

Conclusions

Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25⁺ Tr1 cells represent prospective markers for assessing disease prognosis.

     

PTP1B expression is an independent positive prognostic factor in human breast cancer

Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase that has come into focus as a critical regulator of multiple signaling pathways. The role of PTP1B in breast cancer remains unclear with evidence suggesting that PTP1B can exert both tumor-suppressing and tumor-promoting effects. To better define the role of PTP1B in human breast cancer, and its relationship with HER2, we performed immunohistochemical studies on a large cohort of functionally annotated primary breast cancer specimens. 683 of 1,402 (49 %) evaluable primary breast cancers are positive for PTP1B. There is no statistically significant association between PTP1B expression and age, tumor size, T stage, histologic grade, lymph node status, or histological subtype. Of note, there is no significant association between PTP1B expression and HER2 expression (PTP1B expression 53.1 % in HER2⁺ cancers vs. 47.5 % in HER2^? cancers, p = 0.0985). However, PTP1B expression is significantly associated with estrogen receptor expression (PTP1B expression 50.7 % in ER⁺ cancers vs. 43.1 % in ER^? cancers, p = 0.0137) and intrinsic molecular subtype (PTP1B expression 53.9 % in the luminal B HER2⁺ subtype and 37.9 % in the basal-like subtype). Of note, multivariate analyses demonstrate that PTP1B is an independent predictor of improved survival in breast cancer (HR 0.779, p = 0.006). Taken together, we demonstrate in the largest study to date that (1) PTP1B is commonly expressed in breast cancer, (2) there is no association or functional impact of PTP1B expression in HER2⁺ breast cancer, and (3) PTP1B expression in breast cancer is associated with significantly improved clinical outcome. Until additional studies are performed, caution should be exercised in using PTP1B inhibitors in human breast cancer.     

Infiltrating regulatory T cell numbers is not a factor to predict patient's survival in oesophageal squamous cell carcinoma

CD4/8 status has been previously reported to be a critical factor in the prognosis of oesophageal squamous cell carcinoma (OSCC). In the current study, we investigated the effect of regulatory T cells (Treg; Foxp3+ lymphocytes) on the status of CD4+ and CD8+ T cells in 122 patients with OSCC. Immunohistochemical analysis of Treg was performed using an antibody against Foxp3. The survival rate for low Foxp3 patients was significantly lower than for high Foxp3 patients (P=0.0028 by log-rank test), but Foxp3 status did not significantly correlate with prognosis in CD4/8(+/+) patients or CD4/8(+/-) or (-/+) patients (P=0.5185 and 0.8479, respectively, by log-rank test). We also found that Foxp3 status correlated with CD4/8 status (P=0.0002 by chi2 test) and that the variance of CD8/CD4 ratio in patients with low Foxp3 was larger than in patients with high Foxp3 (P<0.0001 by F-test). Thus, the results do not support the idea that Treg suppress anti-tumour immunity in patients with OSCC. Rather, the CD8/CD4 ratio and CD4/8 status appear to be critical factors in anti-tumour immunity. Furthermore, Treg numbers correlate with both the CD8/CD4 ratio and the CD4/8 status, suggesting that Treg number is not a factor to predict patient's survival in OSCC.     

SR140333 counteracts NK-1 mediated cell proliferation in human breast cancer cell line T47D

Background

It has been demonstrated that certain NK-1 antagonists could reduce proliferation of several cancer cell lines, however, it is unknown whether SR140333 exerts proliferation inhibition in breast cancer cell line.

Methods

Immunohistochemical staining was carried out to investigate the immunolocation of NK-1 in breast cancer tissues and T47D cell line, thereafter, various concentrations of [Sar9, Met(O2)11]substance P and SR140333 were applied alone or combined. MTT assay was applied to detect cytoactivation and coulter counter was to detect growth curve. The Hoechst33258 staining was performed to detect apoptosis.

Results

We found that breast cancer and T47D cells bear positive expression of NK-1. SR140333 inhibited cell growth in a dose dependent manner. Furthermore, SR140333 could counteract [Sar9, Met(O2)11]substance P induced proliferation. Hoechst33258 staining revealed the presence of apoptosis after SR140333 treatment.

Conclusions

Our study demonstrated SR140333 exert proliferation inhibition in breast cancer cell line T47D and indicates NK-1 play a central role in the substance P related cell proliferation in breast cancer.     

Vascular proliferation is a prognostic factor in breast cancer

Angiogenesis is important for the growth and spread of malignant tumors, and anti-angiogenesis treatment is currently being evaluated for breast cancer and other tumors. Although microvessel density is the most commonly used tissue-based marker of tumor associated angiogenesis, it has significant limitations and has not proven effective as a predictive factor in selecting patients for treatment. We here wanted to explore the significance of vascular endothelial cell proliferation in breast carcinoma. We examined microvessel proliferation in breast cancer by dual immunohistochemical staining, using the pan-endothelial marker Factor-VIII combined with proliferation of endothelial cells by Ki-67 expression, in three independent series of breast cancer, including a total of 499 patients and 141 events during follow-up. Common statistical tests of associations as well as univariate and multivariate regression analysis of patient survival were used. By counting vessels with actively proliferating endothelium, we show that microvascular proliferation is a significant predictor of disease progression in breast cancer, especially among high-grade and ER-negative tumors. Our findings indicate that this novel marker of active tumor angiogenesis might be of value in patient management and should be further studied in the context of patient selection for anti-angiogenesis treatment.     

Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)

Introduction

Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1, also known as EBP50 or NHERF) is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity (LOH) at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified.

Methods

To confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA (shRNA) to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses.

Results

We found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G₁-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level.

Conclusion

Our findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities.     

Management of T1a breast cancer

Identification of early-stage breast cancers has increased over the past 2 decades primarily because of mammographic screening. The general guidelines to management of breast cancer may not apply to the smallest of these tumors, as their metastatic potential may be smaller than larger tumors. Tumors < 5 mm (T1a) carry an excellent prognosis, despite a variety of treatment approaches. However, some patients' cancer returns. There appear to be some histologic features that can predict a higher risk of axillary metastases, and therefore, a higher risk of distant metastases. Controversy exists over the extent of treatment, as to whether less than conventional treatment, such as mastectomy, axillary evaluation, and breast-conserving surgery and radiation, can be done. T1a lesions associated with extensive ductal carcinoma in situ and T1a lesions in young patients should be treated with caution if less than conventional breast treatment is to be considered. In older patients with good histologic features, axillary assessment may not be necessary. Very wide excision alone may be appropriate for some patients, but partial breast irradiation is under study and may provide a reasonable compromise. Systemic therapy for node-negative patients is not recommended. Recurrences within the breast occur later in early-stage breast cancers than with extensive-stage breast cancers, requiring annual imaging and evaluation for many years.     

Inhibition of hormone and growth factor responsive and resistant human breast cancer cells by CeReS-18, a cell regulatory sialoglycopeptide

We have previously documented that CeReS-18, a cellregulatory sialoglycopeptide, inhibits the cellular proliferation of normaland transformed cell types from a diverse rangeof species. Most cell types studies exhibit asimilar sensitivity to the reversible but growth inhibitoryeffects of CeReS-18 at 7 × 10^-8 Mconcentration, while at higher concentrations CeReS-18 can elicitcytotoxicity. The present study was conducted to examinethe effect of CeReS-18 on the proliferation ofhuman mammary epithelial carcinoma cells. MCF-7 cells, whichare estrogen receptor positive (ER⁺), and BT-20 cells,which are estrogen receptor negative (ER^-), were utilized.Both cell lines show equal sensitivity to growthinhibition elicited by CeReS-18. Complete cessation of cellcycling was achieved with 7 × 10^-8 MCeReS-18, and the arrest was shown to becompletely reversible. Flow cytometric analysis, performed on CeReS-18treated cells from both cell types, revealed thatthe majority of these cells were arrested inthe G1 phase of the cell cycle. Whencells were treated simultaneously with inhibitor and stimulatoryconcentrations of mitogens such as epidermal growth factor(EGF), basic fibroblast growth factor (b-FGF), estrogen, insulin-likegrowth factors I and II (IGFI and IGFII),no alteration of the inhibitory activity of CeReS-18was observed. CeReS-18 clearly abrogated the mitogenic activitythat these growth factors elicited with human mammarycarcinoma cells.     

Cyclin D1 is necessary for tamoxifen-induced cell cycle progression in human breast cancer cells

Despite the success of tamoxifen in treating hormone-responsive breast cancer, its use is limited by the development of resistance to the drug. Understanding the pathways involved in the growth of tamoxifen-resistant cells may lead to new ways to treat tamoxifen-resistant breast cancer. Here, we investigate the role of cyclin D1, a mediator of estrogen-dependent proliferation, in growth of tamoxifen-resistant cells using a cell culture model of acquired resistance to tamoxifen. We show that tamoxifen and 4-hydroxytamoxifen (OHT) promoted cell cycle progression of tamoxifen-resistant cells after growth-arrest mediated by the estrogen receptor down-regulator ICI 182,780. Down-regulation of cyclin D1 with small interfering RNA blocked basal cell growth of tamoxifen-resistant cells and induction of cell proliferation by OHT. In addition, pharmacologic inhibition of phosphatidylinositol 3-kinase/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 pathways decreased basal cyclin D1 expression and impaired OHT-mediated cyclin D1 induction and cell cycle progression. These findings indicate that cyclin D1 expression is necessary for proliferation of tamoxifen-resistant cells and for tamoxifen-induced cell cycle progression. These results suggest that therapeutic strategies to block cyclin D1 expression or function may inhibit development and growth of tamoxifen-resistant tumors.     

Prolactin acts as a potent survival factor for human breast cancer cell lines

Human breast cancer is the leading cause of cancer death in women from Western societies, and a large study of the epidemiology demonstrated strong associations between human prolactin and risk of breast cancer. Using established models of apoptosis of human breast cancer cell lines, we assessed the role of prolactin in breast cancer cell growth and survival. We showed that prolactin had no effect on the metabolic activity or total cell number of any cell lines. We confirmed endogenous prolactin production by these cells and that the levels varied. In the presence of a prolactin-neutralising antibody, each of the cell lines responded with the induction of apoptosis as opposed to growth inhibition. The sensitivity of the cell lines to the physiological inducer of apoptosis, C2-ceramide, appeared relative to the levels of endogenous prolactin that they contained. We then showed that exogenously added prolactin acted as a potent survival factor against apoptosis in all the cell lines examined. In addition, we demonstrated that a prolactin-neutralising antibody in combination with C2-ceramide caused an anticipated, additive increase in cell death. This study demonstrated that prolactin protects human breast cancer cell lines against apoptosis and this may have important implications for cancer treatment.     

Identification of a protein factor secreted by 3T3-L1 preadipocytes inhibitory for the human MCF-7 breast cancer cell line.

The 3T3-L1 cell line is a preadipocyte cell line derived from the Swiss 3T3 mouse fibroblast cell line. We have compared the effect of 3T3-L1 conditioned medium (3T3-L1 CM) and Swiss 3T3 conditioned medium (3T3 CM) on the growth of normal mouse mammary cells (NMMG) and the human MCF-7 breast carcinoma cell line. 3T3 CM increased the growth of both NMMG and MCF-7 cells by 19 +/- 2% (SD) and 24 +/- 3%, respectively, and increased thymidine incorporation by 74 +/- 4% and 104 +/- 8%, respectively. Conditioned medium from 3T3-L1 cells stimulated the growth of NMMG cells by 64 +/- 2%; in contrast, 3T3-L1 CM inhibited the growth of MCF-7 cells by 36 +/- 1%. In parallel with these growth studies, thymidine incorporation increased by 20 +/- 4% in NMMG cells and decreased by 72 +/- 5% in the MCF-7 cells. Moreover, a similar effect was also noted in NCI H630 colon cancer cells, where 3T3-L1 CM produced a 58 +/- 4% decrease in growth and a 82 +/- 6% decrease in thymidine incorporation. Heating the 3T3-L1 CM at 100 degrees C for 30 min destroyed all inhibitory activity. Several known inhibitory growth factors (fibroblast growth factor, 20 ng/ml; interleukin 6, 1000 units/ml; tumor necrosis factor alpha, 15 ng/ml; transforming growth factor beta, 1 ng/ml) were tested for activity in the MCF-7 cells. Tumor necrosis factor alpha and transforming growth factor beta produced a 97% and 67% inhibition of thymidine uptake, respectively, whereas interleukin 6 and fibroblast growth factor had no effect. Neither transforming growth factor beta nor tumor necrosis factor alpha activity was detectable in 3T3-L1 CM using an enzyme-linked immunosorbent assay. High-performance liquid chromatography fractionation of the 3T3-L1 CM revealed that the inhibitory activity eluted at a molecular weight of 67,000; moreover, silver staining of these eluates on a denaturing polyacrylamide gel revealed that M(r) 69,000 peptide was the predominant protein band in the inhibitory fractions. Thus 3T3-L1 CM stimulates the growth of normal breast epithelial cells and inhibits the growth of MCF-7 breast cancer cells. This inhibitory activity appears to be due to a protein secreted by 3T3-L1 preadipocytes.     

Characterization of insulin-like growth factor 1 receptors (IGF1-R) in human breast cancer cell lines

The binding characteristics of IGF1 on membranes prepared from 5 human breast cancer cell lines were investigated in detail. The presence of one class of high affinity IGF1 binding sites was demonstrated (BT-20: n = 230 fmol/mg protein, Ka = 0.7 nM-1; MCF-7: n = 124 fmol/mg protein, Ka = 1 nM-1; T-47D: n = 61 fmol/mg protein, Ka = 1.1 nM-1; HBL-100: n = 18 fmol/mg protein, Ka = 3.2 nM-1; MDA-MB-231: n = 7 fmol/mg protein, Ka = 2.8 nM-1). Chemical cross-linking of 125I IGF1 to breast cancer cell membranes then sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed one major band of relative molecular weight 130000. The specificity of these receptors was studied: native or recombinant IGF1 had the same potency to inhibit 125I IGF1 binding; IGF2 was able to compete for binding, whereas insulin competed with a potency lower than 1/100 that of IGF1. These characteristics of IGF1 binding sites in breast cancer cell membranes correspond to the previously described binding unit of type I IGF receptors (IGF1-R). Finally we determined that for a routinely used standard assay, it was necessary to incubate for 5 h at 4 degrees C a high amount of membrane protein (400 micrograms) and 200,000 cpm of tracer. Considering the known effect of IGF1 on breast cancer cell multiplication, it is tempting to suggest that this factor might play a major role in the growth of breast cancer: the measurement of IGF1-R, using this standardized method, will give an assessment of these tumors IGF1 sensitivity; it can be performed on the membrane fraction obtained when preparing cytosol for steroid receptor assay.     

Accumulation of regulatory T cells in sentinel lymph nodes is a prognostic predictor in patients with node-negative breast cancer

It has been revealed that sentinel lymph nodes (SLNs) from patients with node-negative breast cancer involve RT-PCR detected micrometastases and isolated tumour cells. However, the prognostic significance of the pathologically undetectable micrometastases is still controversial. In this study, we evaluated Foxp3 positive regulatory T cells (Treg) in SLNs as host-side immune marker that has the potential to detect these micrometastases. In the analyses of training set (n = 30), elevated Treg was strongly associated with the pathologically undetectable micrometastases. In the analyses of validation set (n = 129) in patients with node-negative, relapse-free survival in patients with elevated Treg was significantly shorter than those with lower Treg (p = 0.005). Furthermore, in multivariate analyses, elevated Treg was correlated with relapse-free survival (p = 0.012). Our data indicate that Treg may increase in the microenvironment of SLNs along with pathologically undetectable micrometastases and is a prognostic predictor in patients with node-negative breast cancer.     

Transforming growth factor beta 1 is implicated in the failure of tamoxifen therapy in human breast cancer

Transforming growth factor-beta 1 (TGF-beta 1) is inhibitory for breast epithelial cells in vitro and treatment of breast cancer cell lines with tamoxifen results in a rise in TGF-beta 1 mRNA expression with associated inhibition of cell growth. To study whether these findings apply in vivo we examined TGF-beta 1 mRNA expression in an oestrogen-dependent mouse xenograft system following systemic treatment of the mice with tamoxifen. In agreement with in vitro studies. TGF-beta 1 mRNA expression was sustained at high levels and associated with a reduction in tumour size. A subsequent study of breast tumour tissue from 56 patients demonstrated high levels of TGF-beta 1 mRNA in 45 of the tumours. High expression was found to correlate with premenopausal status, but not with tumour oestrogen receptor content or other parameters. In a subgroup of 11 patients who had received tamoxifen therapy for 3 to 6 months prior to surgery, unexpectedly high levels of TGF-beta 1 mRNA were demonstrated in tumours increasing in size and unresponsive to tamoxifen. Data from this study indicate that in patients with breast cancer, TGF-beta 1 in the tumour may not behave as in vitro and xenograft studies have suggested. We speculate that failure of tamoxifen therapy may be due to failure of the autocrine inhibitory functions of TGF-beta 1 either alone or in combination with paracrine stimulation of stromal cells or angiogenesis and localised immunosuppression. Further studies of active TGF-beta 1, TGF-beta receptors and the interactions with other growth factors will be required to elucidate the precise role of TGF-beta 1 in human breast cancer and in the failure of tamoxifen therapy.     

Na+/H+ exchanger regulatory factor 1 inhibits platelet-derived growth factor signaling in breast cancer cells

Introduction

The gene encoding Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) is a putative tumor suppressor gene that harbors frequent loss of heterozygosity (LOH) and intragenic mutations in breast carcinoma. The exact biologic activity of NHERF1 in mammary glands, however, remains unclear. It was recently proposed that NHERF1 forms a ternary complex with platelet-derived growth factor receptor (PDGFR) and phosphatase and tensin homolog (PTEN), linking NHERF1 suppressor activity to PDGF-initiated phosphoinositide-3 kinase (PI3K)/PTEN signaling.

Methods

The effect of NHERF1 on the kinetics of PDGF-induced Akt activation was determined in cells with varied NHERF1 background. Levels of active Akt in mammary gland of NHERF1 knockout and wild-type mice were compared. We also examined how NHERF1 expression status affects cell sensitivity to PDGFR inhibitor. A plausible connection between NHERF1 and PTEN pathway was explored at the genetic level.

Results

We showed that NHERF1, through its PDZ-I domain, interacts directly with the carboxyl-terminal tail of PTEN. Knocking down NHERF1 expression in Zr75.1 cells markedly delayed the turnover of PDGF-induced phospho-Akt. Conversely, NHERF1 over-expression in MCF10A cells led to accelerated phospho-Akt degradation. The slowed decay of phospho-Akt that resulted from NHERF1 loss was evident in mouse embryonic fibroblasts isolated from NHERF1 knockout mice. In agreement with this, mammary gland tissues from these mice exhibited markedly elevated phospho-Akt. The responses of breast cancer cells to PDGFR inhibition were also altered by changes in NHERF1 expression level. Zr75.1 cells with NHERF1 knockdown were more resistant to STI-571-induced apoptosis than parental cells. Similarly, over-expression of NHERF1 rendered MCF10A cells more sensitive to STI-571. NHERF1-induced apoptotic response relies on an intact PTEN pathway; over-expression of NHERF1 in MCF10A cells with PTEN knockdown did not affect STI-571 sensitivity. It was found that NHERF1 LOH-positive breast cancer cells had reduced NHERF1 expression. Interestingly, these cells more frequently had wild-type PTEN or PI3KCA gene than the LOH-negative lines.

Conclusion

Our data indicate that the interaction of NHERF1 with PTEN counterbalances PI3K/Akt oncogenic signaling and may affect how cells respond to PDGFR inhibition in breast cancer. The dependence of NHERF1 responses on PTEN and genetic segregation of NHERF1 and PTEN (or PI3KCA) alterations suggest that NHERF1 is an active component of the PTEN pathway. Collectively, our study indicates that the biologic activity of NHERF1 in mammary gland is related to PTEN signaling.