Role Of TIP30Deletion In Development Of Breast Cancer In Mice

Background and objectiveBreast cancer is one of the most frequently diagnosed cancers in females worldwide, accounting for23%(1.38million) of the total new cancer cases and14%(458,400) of the total cancer deaths in2008. In general, incidence rates of breast cancer are still increasing year by year, but breast cancer death rates have been decreasing in North America and several European countries over the past25years. However, in many African and Asian countries, both incidence and mortality rates are rising. Although, a great improvement of dignosis and treatment of breast cancer have been made, the mechanism underlining breast cancer remains unclear. Estrogen receptor alpha (ERa) plays important roles in mammary gland development and carcinogenesis. ER positive breast cancer subtype is the most common breast cancer subtype, approximately accounting for60-70%. ER has been a preditive marker for patients’prognosis and a good marker for therapy chosen. ER positive breast cancer has better effect of therapy and prognosis in clinic, however, the machanism of this subtype of breast cancer is still unkown. This study generated an TIP30knockout mouse model and focused on exploring its limited development of ER positive breast cancer.TIP30, also known as CC3, is a30-kDa human cellular protein that was purified as a HIV-1Tat interacting protein and its expression is found altered in human liver, lung, and breast cancers etal. Our previous studies showed that Tip30-deficient mice spontaneously develop tumors in several tissues and mammary preneoplastic lesions, suggesting that TIP30acts as a tumor suppressor. Its tumor suppressor activity is likely due to its actions in multiple cellular processes. TIP30can function as a transcription cofactor to repress expression of genes that are involved in proliferation and apoptosis. TIP30can act as a repressor of ERa-mediated c-Myc transcription in mammary glands and breast cancer cells. Moreover, data about TIP30controling endocytic downregulation of the EGFR signaling pathway in primary hepatocytes、 hepatocellular carcinoma cells and lung cancer cells also have been highlighted recently.Our previous data showed that deleting the TIP30gene in mice leads to ductal hyperplasia in mammary glands early in life and extensive mammary hyperplasia with age. In addition, we also showed that deletion of TIP30in MMTV-Neu mouse can accelerate the onset of mammary tumors and resulted in ER+/PR-mammary tumors. In this study, we generated a TIP30knockout Balb/c mice model without the interference of Neu gene, and found that TIP30deletion in mice developed mammary tumors. We futher analysed the EGFR downstream cell signaling in this model to study the possible machanism of breast cancer development and used human breast cancer cell line to explore the role of TIP30in regulating EGFR degration.Breast cancer has heterogeneous pathologies and molecular profiles and in this respect there are many molecular subtypes of breast cancer. The stem cell model of mammary carcinogenesis provides an explanation for the cellular heterogeneity within tumors, in addition to the phenotypic diversity seen in different subsets of patients. This model predicts that different subsets of mammary carcinoma arise from different cells and/or the particular mutations required to transform these cells. These mutations might result in uncontrolled self-renewal of the cancer stem cell, driving tumorigenicity, and aberrant differentiation, with this last property generating most of the cells within a tumor. In addition, studies in mice have clearly shown that different genes exert their influence in different cell subpopulations. For example, mouse mammary tumour virus (mmTv)-Wnt-1results in tumours that are composed of both luminal and myoepithelial cells and an expansion of the MaSC pool. However, mmTv-Erbb2(Neu) mice can generate luminal cell-restricted tumours and display an expansion of the luminal cell compartment.In our study, we are trying to explore effects of TIP30deletion on mammary stem/progenitor cells pool and their cell fate, which can provide some explainations for our mouse model which spontaneously developed ER positive breast cancers.Part I:Loss of TIP30drives development of mouse breast cancer and activates EGFR downstream signal molecules.Methods1. TIP30knockout Balb/c mice were obtained by backcross TIP30knockout C57J6mice with Balb/c mice. After over7generations continuous passages, we finally established TIp30-/-Balb/c mice.2. After being observed for78weeks, mice were sacrificed for relevant researches. Check the mouse organs such as brain, mammary, lung, etal to see any tumor or desease happened in them. Part of tissues were fixed, dehydrated and embeded in paraffin for futher analysis. Rest of tissues were freshly frozen in liquid nitrogen and were kept in-80℃for longterm storage.3. H&E staining were used to detect the tissue section including the organs and tumors. Slides were examined by two pathologists for tumorgenesis or other aberrant histological changes. After that, incidence of breast carcinoma was caculated.4. Immunohistochemistry (IHC) was performed to analyse expression of CK8, aSMA, pAkt and pERK in mammary gland and mammary tumor.5. Immunofluorescence was used to detect expression of ER and PR in mamary tumor.6. Lentiviruses were generated using shRNA-TIP30and shRNA-CON plasmids and then transfected MCF-7cells following puromycin selection. Transfection efficiencies were analyzed by western-blot.7. Immunofluorescence was used to analyse the colocolization of EGF and EGFR, EGFR and EEA1as well as TIP30and EEA1in MCF-7cells after treated with EGF for indicated times.8. Statistical analysis:Unless otherwise stated, data were expressed as (x±s) and analysed with SPSS13.0. Incidence rate of breast cancer between two groups was analysed by x2test. Comparisons of two groups’or three groups’mean values were analyseed by independent sampe t test or One-way ANOVA followed by LSD or Dunnett multiple comparison test. P-Values were considered to be significant at <0.05.Results1. Knockout of TIP30results in spontaneous mammary tumor in Balb/c mice.After18months observeation,47female mice reached the analysis end point.8of28Tip30-’-mice were found having mammary tumor, but no tumor found in19Tip30+/+mice (0%). Incidence rate of mammary tumor in Tip30-/-mice (28.6%) is significantly higher than Tip30+/+mice (0%), x2=6.269, P=0.012.2. Mammary tumor induced by TIP30deletion mice were all ER/PR postive luminal type.Mammary tumor tissue section were stained with K8and by IHC and results show edthat all the tumors are K8strongly posive, but aSMA negative, which implied that mammary tumors found in Tip30-/-mice were Luminal subtype. Then we used immunofluorescence to dectect ER/PR expression and found that all tumor sections are ER postive and PR positive. All these data indicated that Tip30-’-mice spontaneous developed ER/PR positive, Luminal type breast cancer.3. TIP30deletion leads to EGFR pathways such as MAPK and PI3K activation.IHC was used to dectect pErkl/2and pAkt expression in mammary gland and mammary tumor sections. Positive rates of pErkl/2and pAkt in Tip30-/-mice mammary gland were27.83±8.46%and30.83±6.65%(n=5), which were significantly higher than12.58±5.87%and14.94±5.77%in Tip30+/+mice respectively, P values were0.02and0.011. Moreover, positive rates of pErkl/2and pAkt in Tip30-/-mice mammary tumor were51.68±8.57%and56.08±8.44%, significantly higher than mammary gland, P values were0.002and0.001 respectively.4. TIP30knockdown could prolong activation of EGFR downstream signal molecules of mamary tumor cells induced by EGF.4.1TIP30-SH1and TIP30-SH2transfection respectively downregulated the expression of TIP30in MCF-7cells.After transfection and anibiotic selection, relative TIP30expression of TIP30-SHland TIP30-SH2cells are13.12±3.95%and11.54±2.68%(n=3), significantly lower than shRNA-CON (84.3±8.66%, n=3) respectively, both of P values less than0.001. In general, TIP30expression of TIP30-SH1and TIP30-SH2cells were downregulated by71.18%and72.76%respectively, which implicated that the transfecting efficience was well.4.2TIP30could promote degration of EGFR induced by EGFAfter treated with EGF, EGF and EGFR expressions were analysed by immunofluorescence at indicated times1Omin,1h,4h. We found that both intencity of EGF and EGFR fluorescence were no difference between TIP30-SH and shRNA-CON cells at10min and1h after treated with EGF, and the Pearson’s colocalization of EGF and EGFR were not different either. However, at4h after treated with EGF, both intencity of EGF and EGFR fluorescence in TIP30-SH cells were1.19±0.18and1.33±0.13, significantly higher than0.68±0.08and0.76±0.10in shRNA-CON cells respectively (1=4.563and6.132, P=0.01and0.004); at the same time, the Pearson’s colocalization of EGF and EGFR in TIP30-SH cells was significantly higher than shRNA-CON cells (35.07±5.33vs.10.76±2.28; t=7.270, P=0.002).4.3TIP30knockdown could stagnate EGFR in early endosomes.2h after treated with EGF, EGFR and early endosome marker EEA1expressions in cells were analysed by immunofluorescence. The Pearson’s colocalization value of EGFR and EEA1in TIP30-SH was56.39±14.08%, significantly higher than27.31±9.12%of shRNA-CON.4.4TIP30could assemble in early endosome and regulate EGFR degration.We also used immunofluorescence to dectect colocalization of TIP30and EEA1, and found that TIP30are colocalized with EEA1at10min after shRNA-CON cells treated with EGF.Conclusion1. TIP30knockout Balb/c mice can spontaneously develop mammary tumor.2. All the mammary tumor developed by TIP30knockout Balb/c mice are ER/PR+Luminal type.3. TIP30deletion results in EGFR downstream pathways such as MAPK and PI3K activation.4. Inhibition of Tip30could delay EGF-induced degradation of EGFR, thus prolong EGFR downstream signal molecules and promote the proliferation of breast cancer cells.Part Ⅱ:TIP30deletion promotes mouse mammary stem/progenitor cells expansion and regulates progenitor cell fateMethods1. Gained Tip30-/-Balb/c and Tip30-/-Neu+/+FVB mice by crossbreeding. After over7generations continuous passages, we finally established TIP30knockout mice with different backgrounds.2.5-6month old mouse mammary tissues were dissociated mechanically and enzymatically. Finally the mammary cell suspensions were obtained.3. Mammosphere culture was used to detect the mammosphere forming capacity of mammary cells from different genotype mice. Caculated the mammosphere’s number and size under microscope.4. Colony forming assay in vitro was performed to analyse the colony generation capacity of mammary cells from different genotype mice. Colony number and size were caculated under microscope.5. Flow cytometry was used to analyse the population of CD24lighCD49flow (MRUs) and CD24HighCD49flow (Ma-CFCs) in mammary cells from different genotype mice, then analyse the subpopulation of Scal+and Seal-in CD24HighCD49flow population. 6. Flow cytometry was used to sort out CD24HighCD49flow population which were gown in vitro using colony forming assay, then were stianed with ER/PR by immunofluorescence.7. Immunofluorescence was used to detect ER/PR expression of the mammary glands.8. QPCR was used to the mRNA levels of factors which related to mammary stem/progenitor cells fate decision.9. Used Western-blot to detect FOXA1and RANKL expression of mammary tissues.10. Lentiviruses were generated using shRNA-FOXAl and shRNA-CON plasmids and then transfected TIP30-/-cells following puromycin selection. After the transfection efficiencies were confirmed by western-blot. The cells were grown in vitro to form colonies and then stained with ER.11. Statistical analysis:Unless otherwise stated, data were expressed as (x±s) and analysed with SPSS13.0. Comparisons of two groups’or three groups’mean values were analyseed by independent sampe t test or One-way ANOVA followed by Dunnett multiple comparison test. P-Values were considered to be significant at≤0.05.Results1. Loss of TIP30can promote mammary stem/progenitor cells expansion.1.1TIP30deletion could increase mammosphere forming capacity.After9d mammosphere culture in serum free medium, mammosphere forming number of Tip30-/-Balb/c was31.75±5.85/5000cells seeded, significantly higher than19.75±4.79/5000cells of7ip30+/+Balb/c, t=3.174, P=0.019. We found a similar result in Neu+/+FVB background mice. Tip30-/-Neuw+/+FVB mice had more mammospheres than Tip30+/+Neu+/+FVB mice (31.25±5.56/5000cells vs.17.5±4.04/5000cells), t=4.001, P=0.007.We also measured the size of mammospheres and found that mammospheres formed by by mammary cells of Tip30-’’Balb/c mice had larger size than that in Tip30+/+Balb/c mice.(156.80±29.28μm vs.97.59±20.66μm; t=3.305, P=0.016). But we can’t find any difference in size between Tip30-/-Neu+/+FVB (128.29±27.83μm) and Tip30+/+Neu+/+FVB mice(105.53±14.19μm),t=1.457,P=0.195.1.2TIP30deletion could enhance colony forming capacity in vitro.2000Mammary cells/well from different mice were seeded on matrigel precoated well for10d.The colony number of Tip30-/-Balb/c(33.33±5.13)was significantly higher compaired with Tip30+/+Balb/c mice(19.00±6.00),t=3.144,P=0.035. Moreover,mammary cells from and Tip30-/-Neu+/+FVB also formed more colonies than those from Tip30+/+Neu+/+FVB respectively(33.00±5.29vS.18.00±4.36),t=3.709,P=0.019.When measuring the size of colonies,we found that Tip30-/-Balb/c(137.9±16.17μm) has larger colony than Tip30+/+Balb/c(90.79±22.89¨m),t=2.914,P=0.044.However, there was no difference in size between Tip30-/-Neu+/+FVB(126.68±25.42μm)and Tip30+/+Neu+/+FVB mice(97.41±13.13Um),t=1.772,P=0.151.1.3TIP30deletion can promote MRUs expansion.Flow cytometry analysis showed that MRUs population of Tip30-/-Balb/c accouting for2.83±0.51%was significantly higher than Tip30+/+Balb/c which wasl.27±0.51%, t=3.739.P=0.02.We got a silimar result which showed that TIP30deletion can significantly increase the MRUs population from1.30±0.60%to6.73±0.85%in Neu+/+FVB mice,t=9.042,P=0.001.1.4TIP30deletion can increase Ma-CFCs.Flow cytometry analyse also showed that Ma-CFCs population of Tip30-/-Balb/c was3.23±0.49%,significantly higher than0.87±0.38%of Tip30+/+Balb/C,t=6.592, P=0.003.For Neu+/+FVB mice,TIP30deletion resulted in Ma-CFCs population increasing from1.43±0.31％to3.07±O.76&,t=3.465,P=0.026.2.TIP30deletion in mice can increase ER+mammary progenitor cells.We used balb/c and Neu+/+FVB mice for further analysis.Sca-1was used for discriminate mammary progenitors cells to two suppoulations,CD24HighCD49flow Scal+and CD24HighCD49flowScal-which represent ER+and ER-progenitor cells respectively.Flow cytometry analysis showed that TIP30deletion can significantly increase the CD24HighCD49flowScal+population from17.73±1.76%to30.67±2.45%, t=7.415, P=0.002. In addition, Tip30-/-Neu+/+FVB mice also had more ER+progenitor cells compared with Tip30+/+Neu+/+B mice (23.80±5.27%vs.13.57±2.70%), t=2.994, P=0.04。3. TIP30deletion can predispose mammary progenitor cells to ER+cell fate.Flow cytometry was used to sort out the mammary progenitor cell population (CD24HighCD49flow), and the cells were plated on the matrigel precoated well and cultured to form colonies then stained with ER/PR by immunofluorescence. We found that colonies formed by Tip30-’-Balb/c mice have more ER positive cells than colonies formed by Tip+/+Balb/c mice (45.35±7.80%vs.31.77±6.38%), t=2.698, P=0.036. But we can’t find difference in number of PR positive cells in colonies form by Tip30-/-and Tip30+/+Balb/c mice (38.70±8.45%vs.33.76±6.33%), t=0.936, P=0.385.To further comfirm the influence of TIP30deletion on ER positive cell differentiation of mammary cells, we checked the ER/PR expression of mouse mammary gland by immunofluorescence. Tip30-/-Bdlb/c mammary glands had more ER positive cells than Tip30+/+Balb/c mammary gland (21.13±3.59%vs.13.00±3.716%, t=3.15, P=0.020). However, there was no difference of PR positive cells between Tip30-/-Balb/c and Tip30+/+Balb/c mammary glands.4. Loss of TIP30upregulated FOXA1expression of mammary glands.We next measured some signaling pathway (Notch, Hedgehog, Wnt) or genes (GATA3、FOXA1) related to cell fate decision at mRNA levels. Only FOXA1mRNA level was found markedly upregulated in Tip30-/-Balb/c mouse mammary glands compared with Tip30+/+Balb/c mice (3.05±0.90vs.1.00±0.17), t=3.879, P=0.018. We further analysised the protein level of FOXA1. The relative expression of FOXA1in Tip30-/-Ba\b/c mammary glands was66.24±5.93%, significantly higher than40.27±8.18%in Tip30+/+Balb/c mammary glands, t=4.454, P=0.011.5. Down regulation of FOXA1in Tip30-/-mammary cells decreased ER+cell differentiation.To determine whether upregulation of FOXA1in Tip30-/-mammary cells was responsible for more differentiation of ER+cell than Tip30+/+mammary cells. We harvested the mammary cells from Tip30-/-balb/c mice, and knocked down the FOXA1expression. After confirmation of knockdown of FOXA1by western blot, cells were grown in matrigel to form colonies which were then stianed with ER. The percentage of ER+cells in colonies formed by FOXA1-SHland FOXA1-SH2cells were31.00±2.85%and29.52±5.67%respectively, both of which were significantly lower than39.40±6.16%in colonies formed by shRNA-CON; P values were0.042and0.018respectively.6. TIP30deletion upregulated RANKL expression of mammary glands.To determine why TIP30deletion can result in expansion of mammary stem/progenitor cells, expression of RANKL was measured by western blot. We found that the levels of RANKL were markedly increased in Tip30-’-Ba\b/c mammary glands than in Tip30+/+Balb/c mammary glands (16.91±1.13vs.7.94±4.47, t=3.370, P=0.028) which indicated that TIP30deletion may promote mamamry stem/progenitor cells expansion through upregulaion of RANKL in a paracrine manner.Conclusion1. TIP30deletion can promote mammary stem/progenitor cells expansion in mice.2. TIP30deletion can increase ER positive progenitor cells, and predispose mammary progenitor cells to ER+cell fate through upregulation of FOXA1.3. TIP30deletion may induce secretion of RANKL from hormone receptor positive cells and promote the mammry stem/progenitor cells expansion by paracrine way.