TITLE of PART I: MICRORNA-205 IS A TUMOR SUPPRESSOR IN BREAST CANCER Major Professor: Dr. Yinyuan Mo MicroRNAs as a class of novel negative regulators have drawn intensive attention since their existence in mammals was discovered in 2001. As endogenous small non-coding RNAs, miRNAs are capable of silencing gene expression at the post-transcriptional level by translation blocking or/and mRNA degradation. MicroRNA has been reported to involve in diverse biological events. Although miRNA deregulation has been observed in many types of cancers including breast cancer, only few of these de-regulated miRNAs have been confirmed to be related with breast cancer progression. This study is to investigate the association of miRNAs with the development of breast cancer. In this study, we report that mir-205 is significantly under-expressed in breast tumors compared to the matched normal breast tissues. Similarly, breast cancer cell lines including MCF-7 and MDA-MB-231 express a much lower level of mir-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of mir-205 significantly inhibits cell proliferation and anchorage-independent growth as well as cell invasion. Furthermore, mir-205 was shown to suppress lung metastasis in a mouse model. Finally, western blot combined with the luciferase reporter assays demonstrated that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets of mir-205 and such mir-205-mediated suppression is likely through the direct interaction with the putative mir-205 binding site in the 3'-untranslated region (3-UTR) of ErbB3 and VEGF-A, suggesting that down-regulation of ErbB3 and VEGFA likely contributes to the suppression role of mir-205 in breast cancer cells. Together, these results suggest that mir-205 is a tumor suppressor in breast cancer. Given that mir-205 is downregulated in breast tumor specimens and breast cancer cell lines, it would be interesting to determine how mir-205 is regulated in cancer cells. Supprisingly, we found that agents which have been previously shown to induce other miRNAs, such as p53 induction, the phenolic compound kaempferol, hypoxia, and ROS stress have little effect on mir-205 expression, whereas de-methylation and UV treatment only a moderate effect on mir-205. Therefore, our studies indicate that mir-205 is a tumor suppressor in breast cancer progression, however, regulation of mir-205 still remains to be elucidated. Knowledge gained from these studies will provide more comprehensive understanding of how miRNAs are associated with tumor initiation and progression in breast cancer and how miRNAs are de-regulated during cancer development, and as a result, mir-205 may serve as a novel target for cancer therapy. TITLE of PART II: REGULATION OF ESTROGEN-INDEPENDENT GROWTH BY MICRORNAS Major Professor: Dr. Yinyuan Mo Tamoxifen (TAM) has proven to be effective in the treatment of breast cancer. The primary target of TAM in vivo is estrogen receptor (ER), and thus levels of ER expression are the best predictors of benefit from TAM. Unfortunately, over 30% of ER-positive tumors fail to respond to TAM therapy; moreover, those breast tumors that initially respond to TAM will frequently develop resistance to the treatment. Although it is believed that this is likely due to the activation of estrogen-independent signaling, the precise molecular mechanism is not fully understood. This poses a significant challenge to the determination of treatment options for ER positive breast tumors. In this study, we performed in vivo selection experiments using the breast cancer ER positive MCF-7 cells infected with a pooled human microRNA library. It is well known that MCF-7 cells do not grow tumors in a xenograft mouse model unless estrogen is provided. We found that the MCF-7 cells infected with the microRNA library, but not the vector control, did grow tumors, suggesting that certain microRNAs are able to support the estrogen-independent growth. To characterize these microRNA-induced tumors, we recovered cells from the tumors in cell culture and found that they are more resistant to TAM than the MCF-7 cells with vector control. Moreover, they are able to grow in estrogen-free medium. Further analysis revealed that these recovered cells are ER positive with highly activated pAkt. By genomic DNA based real-time PCR, we identified that 6 miRNAs have been enriched in the tumor genome. Co-infection of these 6 enriched miRNAs is able to confer MCF-7 cells estrogen-independent growth and tamoxifen resistance. Further analysis indicated that mir-101 alone is sufficient to induce in vitro estrogen-independent growth and tamoxifen resistance. Together, these results suggest that microRNAs are important players in regulating the estrogen-independent growth and thus, identifying such microRNAs and understanding the underlying mechanisms will provide new insight into estrogen-independent tumor growth and TAM resistance, and thus, these microRNAs may serve as novel targets for breast cancer therapy.

Resistance to antiestrogen therapy remains a significant problem in breast cancer. Low expression of inhibitor of growth 4 (ING4) in primary tumors has been correlated with increased rates of recurrence in estrogen receptor-positive (ER+) breast cancer patients, suggesting a role for ING4 in ER signaling. This study provides evidence that ING4 inhibits ER activity. ING4 overexpression increased the sensitivity of T47D and MCF7 ER+ breast cancer cells to hormone deprivation. ING4 attenuated maximal estrogen-dependent cell growth without affecting the dose-response of estrogen. These results indicated that ING4 functions as a noncompetitive inhibitor of estrogen signaling and may inhibit estrogen-independent ER activity. Supportive of this, treatment with fulvestrant but not tamoxifen rendered T47D cells sensitive to hormone deprivation as did ING4 overexpression. ING4 did not affect nuclear ER alpha protein expression, but repressed selective ER-target gene transcription. Taken together, these results demonstrated that ING4 inhibited estrogen-independent ER activity, suggesting that ING4-low breast tumors recur faster due to estrogen-independent ER activity that renders tamoxifen less effective. This study puts forth fulvestrant as a proposed therapy choice for patients with ING4-low ER+ breast tumors.