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Tip30 deletion in MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen receptor-positive and progesterone receptor-negative mammary tumors - PubMed

️Fri Jan 01 2010

Tip30 deletion in MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen receptor-positive and progesterone receptor-negative mammary tumors

Chengliang Zhang et al. Cancer Res. 2010.

Abstract

Estrogen receptor-positive and progesterone receptor-negative (ER+/PR-) breast cancers account for 15% to 25% of all human breast cancers and display more aggressive malignant characteristics than ER+/PR+ cancers. However, the molecular mechanism underlying development of ER+/PR- breast cancers still remains elusive. We show here that Tip30 deletion dramatically accelerated the onset of mammary tumors in the MMTV-Neu mouse model of breast cancer. The mammary tumors arising in Tip30(-/-)/MMTV-Neu mice were exclusively ER+/PR-. The growth of these ER+/PR- tumors depends not only on estrogen but also on progesterone despite the absence of detectable PR. Tip30 is predominantly expressed in ER+ mammary epithelial cells, and its deletion leads to an increase in the number of phospho-ERα-positive cells in mammary glands and accelerated activation of Akt in MMTV-Neu mice. Moreover, we found that Tip30 regulates the EGFR pathway through controlling endocytic downregulation of EGFR protein level and signaling. Together, these findings suggest a novel mechanism in which loss of Tip30 cooperates with Neu activation to enhance the activation of Akt signaling, leading to the development of ER+/PR- mammary tumors.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest.

No potential conflicts of interest were disclosed

Figures

**Figure 1**
*Tip30* deletion significantly accelerates the onset of mammary tumors in MMTV-Neu mice. *Neu+*/*Tip30^+/+* (*n =* 10), *Neu+*/*Tip30^+/−* (*n =* 15) and *Neu+*/*Tip30^−/−* (*n =* 10) female mice were monitored weekly for a period of 75 weeks and sacrificed at the endpoint or when tumor volume reached 0.5 cm³. A, Kaplan-Meier analysis of survival as function of palpable tumor. The data were plotted as percentage of tumor-free animals against the time in weeks. P ≤ 0.0001; log-rank test. B, Representative hematoxylin and eosin (H&E) stained mammary tumors arising in *Neu+*/*Tip30^−/−* mice. A poorly differentiated adenocarcinoma with solid growth pattern (B1); A moderately differentiated adenocarcinoma with glandular growth pattern (B2); A pulmonary metastasis (B3). Scale bar, 50 μm. C, Representative immunohistochemical staining of mammary tumors for K8 (brown staining indicates presence of K8) and αSMA (lack of brown staining indicates lack of αSMA). Scale bar, 10 μm.

**Figure 2**
Mammary tumors arising in *Neu+*/*Tip30^−/−* mice are exclusively ER+/PR−. A-C, Representative immunofluorescent staining of ERα (red), PR-A (green) and PR-B (green) in the positive control uterus (A) and mammary tumors arising in *Neu+*/*Tip30^−/−* mice (B) or *Neu+*/*Tip30^+/+* mice (C). Tumor sections were stained with anti-ERα, anti-PR-A (hPRa7) or anti-PR-B (hPRa6) specific antibodies, followed by counterstaining with DAPI. Scale bar, 10 μm.

**Figure 3**
Growth of ER+/PR− tumors arising in *Neu+*/*Tip30^−/−* mice depends upon estrogen and progesterone. A, Two ER+/PR− mammary tumors from *Neu*+/*Tip30^−/−* mice were minced and inoculated subcutaneously (s.c.) in the front flanks of ovary-intact (*n =* 16) or ovariectomized (*n =* 9) mice. The graph represents the measurements of tumors by the end of three months after transplantations or when the tumor volume reaches 1 cm³. P = 0.012. B, Two ER+/PR− mammary tumors from *Neu*+/*Tip30^−/−* mice were minced and inoculated s.c. in the front flanks of ovariectomized mice supplemented with placebo (*n =* 10), estrogen (E2, *n =* 6), progesterone (P4, *n =* 6) or E2 plus P4 (*n =* 7) pellets. The graphs show the measurements of tumor volumes by the end of three months after transplantations or when the tumor volume reaches 1 cm³. P = 0.039 (Placebo vs E2 + P4). Note that tumor growth in two mice of the placebo group was independent of ovarian hormones. C, Growth of ER+/PR− tumors after being treated with saline/ethanol vehicle or RU486. Two ER+/PR− tumors arising in *Neu+/Tip30^−/−* female mice were minced and inoculated s.c. to nude mice. After transplanted tumors reached approximately 0.5 cm in diameter, mice were divided into two groups to be treated with either RU486 (6.5 mg/kg body weight) or saline/ethanol vehicle solution s.c. daily for 7 days. Tumor size was measured by caliper (length and width) for another 7 days. Tumor increase rate was calculated by comparing tumor volume (1/2 × length × width²) before and after treatment. P = 0.025. D, Primary tumor cells derived from two ER+/PR− tumors (T1 and T2) were serum-starved and cultured in the presence or absence of 10uM MG132 for 6 hours. Cell lysates were subjected to Western blot analysis with the anti-PR antibody hPRa7 that detects both PR-A and PR-B. K8 is degraded by proteasomes (45) and was blotted as a positive control for MG132 inhibition.

**Figure 4**
Representative immunohistochemical staining of p-ERα in mammary glands and mammary tumors from *Neu+*/*Tip30^−/−* and *Neu+*/*Tip30^+/+* mice. A, Representative immunofluorescent double staining of mammary gland and tumor sections from a *Neu+*/*Tip30^+/−* mouse for ERα (red) and β-Gal (green), followed by counterstaining with DAPI (blue). Scale bar, 10 μm. B, Representative immunohistochemical staining of p-ERα in 2-month-old and 12-month-old mammary glands and mammary tumors from *Neu+/Tip30^−/−* and *Neu+/Tip30^+/+* mice. As a negative control, a uterus section was stained without using the primary antibody (anti-pERα). Scale bar, 10 μm. C, Data represent means ± SEM of the percentage of p-ERα positive cells in the mammary glands and tumors derived from *Neu+/Tip30^−/−* and *Neu+/Tip30^+/+* mice. Positive p-ERα cells were counted in the sections of mammary glands and tumors derived from three mice of each genotype (randomly selected fields per section). 50 cells were counted per field and 10 fields were counted per mouse.

**Figure 5**
Representative immunohistochemical staining for p-Akt in mammary tumors and mammary glands from *Neu+*/*Tip30^−/−* and *Neu+*/*Tip30^+/+* mice. A, Representative immunohistochemical staining of p-Akt in mammary glands. Staining of p-Akt in 2-month-old mammary glands ranges from negative to weak (A1 and A2) and is more intense in 12-month-old mammary glands (A3 and A4, intermediate and strong, respectively). Scale bar, 10 μm. B-D, Data represent means ± SEM of the percentage of cells that were stained positive or negative for p-Akt in 2-month-old (B) and 12-month-old (C) mammary glands and tumors (D) derived from *Neu+*/*Tip30^−/−* and *Neu+*/*Tip30^+/+* mice. Fifty cells were counted per field and 10 fields were counted per mouse. Data were analyzed by two-tailed t test.

**Figure 6**
Deletion of *Tip30* in MECs leads to delayed EGFR degradation. A, *Neu+*/*Tip30^−/−* and *Neu+*/*Tip30^+/+* mammary tumor cells were incubated with 100 ng/ml of EGF for 1 hour on ice followed by washing with cold PBS and incubating in serum-free medium containing cycloheximide (20 μg/ml) for the indicated times. Whole cell lysates were blotted with the indicated antibodies. B, Quantification of EGFR protein levels in (A) using Odyssey 2.1 software. C, primary *Tip30^+/+* and *Tip30^−/−* MECs were subjected to EGFR internalization analysis. Representative confocal microscopy images show the localization of EGFR (red) in endosomes after two hours of Alexa⁴⁸⁸-EGF (green) internalization. Results are typical and representative of three experiments on primary cells from two mice of each genotype. Boxed areas are magnified. Representative cells are outlined in white. The colocalization of EGF and EGFR (yellow) in *Tip30*^−/− cells is indicative of delayed endocytic degradation of EGFR; the nucleus was stained with DAPI (Grey). Scale bar, 10 μm. D, Quantitative analysis of EGF and EGFR colocalization. Twenty cells in each group were analyzed using MBF_imageJ. Pearson’s colocalization coefficients were calculated and converted to percentages. P = 0.035.

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Tip30 deletion in MMTV-Neu mice leads to enhanced EGFR signaling and development of estrogen receptor-positive and progesterone receptor-negative mammary tumors - PubMed