Most breast cancer-related deaths are due to distant metastasis that occur years after successful tumor removal and treatment. Tumor metastasis is a multi-step process in which primary tumors lose cell polarity and undergo dissemination by Epithelial-Mesenchymal Transition (EMT). The disseminated tumor cells circulate throughout the body and often remain in a dormant state after reaching distant organs. Once the microenvironment becomes suitable, the cells undergo Mesenchymal-Epithelial Transition (MET) and exit dormancy, which results in macrometastases. Previous studies show that a transcription factor Twist1 plays a key role in inducing EMT and promoting tumor metastasis. However, the molecular mechanisms of metastasis are not well understood due to the lack of an in vivo mouse model that can monitor disseminated tumor cells. Therefore, we established a Twist1-inducible breast cancer mouse model that utilizes fluorescent markers to visualize and characterize disseminated tumor cells at various stages of metastasis.

During metastasis, carcinoma cells acquire invasive abilities to invade locally and to spread to distant organs. The ability to degrade extracellular matrix (ECM) to breakthrough the basement membrane, undergo local invasion, intravasation, and extravasation is crucial for tumor cells to metastasize. Tumor cells acquire ECM degradation ability through the formation of membrane protrusions termed invadopodia to localize various proteases to cell-matrix contact points. Invadopodia are specialized actin-rich cell protrusions that contain a number of actin regulatory proteins including cortactin, Arp2/3, and N-WASP. The matrix degradation activity of invadopodia has been associated with a number of proteases, including MMPs and membrane type MMPs. Inhibiting essential invadopodial components, including cortactin, Tks5, or MT1-MMP, inhibited breast cancer metastasis in vivo, supporting a critical role for invadopodia in tumor metastasis. Our lab previously discovered transcription factor TWIST1 promotes tumor metastasis by inducing invadopodia-mediated matrix degradation and tumor invasion. Additionally, TWIST1 induces the expression of Fibroblast Activation Protein (FAP), a membrane-associated serine protease that have been associated with invadopodia. Furthermore, FAP is essential for focal ECM degradation and is required for TWIST1-mediated breast cancer metastasis in vivo, however, the protease activity of FAP is not required for regulating invadopodia function. This dissertation characterized the role of FAP in invadopodia function and metastasis in two new cell lines and found that FAP deficient cells have less percentage of cells forming invadopodia. Further study suggests that FAP is not required for invadopodia initiation but may be required for invadopodia stabilization. To better understand the role of FAP at invadopodia, an immunoprecipitation followed by mass spectrometry was performed to identify FAP interacting proteins. We found FNDC3B, a transient interactor of FAP, that localizes to invadopodia and regulates invadopodia function. Despite FNDC3B deficient cells sharing similar degradation defect as that of FAP, FNDC3B is not required for invadopodia formation. The findings suggest FAP and FNDC3B have separate functions at invadopodia. We believe that FNDC3B helps promote MT1-MMP vesicle transport to invadopodia while FAP proper MT1-MMP localization once it is at invadopodia. Further studies must be undertaken to better understand the dynamics of FAP and FNDC3B at invadopodia.

Breast cancer metastasis, the dissemination of primary tumor cells to distant organs, is the leading cause of death among breast cancer patients. During metastasis, tumor cells must invade through the basement membrane and local tissues, intravasate into the circulatory system, travel to a distant organ, extravasate into the new tissue, and establish micrometastases, which eventually become secondary tumors. The basic helix-loop-helix transcription factor Twist1 is expressed in a variety of aggressive cancers, and is essential for metastasis to take place. Twist1 activates a developmental program known as the Epithelial-Mesenchymal Transition (EMT), during which tumor cells lose their cell-cell junctions and become more migratory and invasive. Twist1 endows tumor cells with an increased invasive capacity by inducing formation of subcellular structures known as invadopodia. Located on the ventral surface of invasive cells, invadopodia are actin-rich protrusions of the cell membrane that recruit proteases to points of cell-matrix contact, and thus degrade the surrounding extracellular matrix (ECM). It was found that Twist1-mediated formation of invadopodia is required for metastasis to take place, but the precise role that Twist1 plays in invadopodia assembly was not completely understood. This dissertation presents data demonstrating that Twist1 is involved in regulation of a serine protease known as Fibroblast Activation Protein alpha (FAPα). FAPα is located at invadopodia, and while not required for EMT or cellular migration, is essential for metastasis to take place. Further investigation found that FAPα expression is required for ECM degradation, but its protease activity is not. Although ECM degradation is impaired when FAPα expression is suppressed, early stages of invadopodia formation still take place, suggesting that FAPα may be involved in the later steps of invadopodia assembly, when proteases are recruited. Indeed, results show that the matrix metalloprotease MT1-MMP is mislocalized in the absence of FAPα expression, providing additional evidence that the role of FAPα at invadopodia is more structural than proteolytic. Further studies must be undertaken to examine how FAPα is trafficked to invadopodia, and which domain of the protein is important for its function.

Metastasis is a complex process in which cancer cells spread from a primary tumor site to invade surrounding tissue, enter the bloodstream, and propagate into distant organs. One of the methods in which metastasis occurs is through Epithelial-to-Mesenchymal Transition, where the extracellular matrix is degraded and restructured; consequently, this enhances cellular migration, invasion, and ultimately, metastasis. A key component of this process is the formation of actin-rich protrusions called invadopodia that serve to break down the extracellular matrix, thereby facilitating cancer invasion. Meanwhile, the E3 ubiquitin ligase ITCH, a key regulator in ubiquitination, has an unexplored role in invadopodia formation. My project thus aims to elucidate ITCH's contribution to tumor invasion via invadopodia-mediated degradation across various cancer cell lines.

I initially focused on ITCH’s role in invadopodia-mediated degradation in the MDA-MB-435 (melanoma) and MDA-MB-231 (triple-negative breast cancer) cell lines, whose ability to form invadopodia was unknown. Degradation and immunofluorescence assays were conducted at various time points and cell confluences. Based on these results, the analysis was expanded to more aggressive cell lines: A673 (Ewing sarcoma), EWS-502 (Ewing sarcoma), TC-32 (neuroectodermal), and SK-N-MC (neuroblastoma). Degradation assays were repeated at different time points and concentrations. Promising results led to further investigation of A673 and EWS-502 by downregulating ITCH expression via lentivirus-mediated shRNA gene knockdown and repeating the degradation assays. Despite comprehensive analysis, the results were inconclusive. However, this project provided preliminary data and established a foundation for future studies to determine the precise role of ITCH in invadopodia formation and degradation, particularly in the A673 and EWS-502 cell lines.

Breast tumors are often identified based on their apparent hardness compared to normal breast tissue, and in breast cancer patients an increase in tissue rigidity is often correlated with an increase in metastasis. When human mammary epithelial cells are grown in 3D culture with the matrix stiffness of breast tumors, they develop a more malignant phenotype. The link between increased tissue rigidity and invasion and metastasis at the molecular level is not well described and is the focus of this dissertation. The transcription factor Twist1 is a key regulator of metastasis through its ability to induce Epithelial-Mesenchymal Transition (EMT), a developmental program also used by cancer cells to invade and metastasize. Previous research in the Yang lab has described a mechanosensing pathway in which Twist1 nuclear translocation is stimulated by increases in matrix rigidity. This nuclear translocation is controlled by phosphorylation of Twist1 on a tyrosine residue which decreases the interaction between Twist1 and its cytoplasmic binding partner G3BP2, allowing Twist1 to enter the nucleus where it can induce EMT. Tyrosine kinase 2 (Tyk2) was identified in a knockdown screen, where loss of Tyk2 was shown to increase nuclear Twist at lower rigidities. This dissertation characterizes the role of Tyk2 in regulating the Twist1-mechanosensing pathway. I describe how Tyk2 is affecting known components of the Twist1-mechanosesning pathway. I also show that Tyk2 activity and subcellular localization are regulated by rigidity. Tyk2 localizes to the plasma membrane at low rigidities and becomes cytoplasmic at high rigidities. This localization change for Tyk2 is recapitulated in human breast tissue samples. Tyk2 is membranous in normal breast tissue and low grade ductal intraepithelial neoplasia (DIN) lesions, which have softer ECM signatures. In ductal carcinoma in situ (DCIS) and breast tumors samples, which have stiffer ECM signatures Tyk2 is cytoplasmic. These findings indicate that Tyk2 is a negative regulator of Twist1 translocation in response to stiffness. This work also reveals a novel role for Tyk2 loss of function in breast cancer invasion and metastasis.

The discrepancy between perceived spectrum shortage and the actual availability by measurements motivates the use of cognitive radio concepts. In this approach the radio locates and then transmits in unused or lightly used bands. If a wideband digital approach (on the order of GHz) to channel selection is taken to provide the necessary radio flexibility, there is a stringent dynamic range and speed requirement in the analog to digital conversion process. This arises from the large interfering signals which are effectively in-band since they are not removed by analog pre-filters. Given this extremely challenging wideband dynamic range goal, a fundamentally different mixed signal architecture has been pursued which is based on time domain signal cancellation. The objective of this thesis is therefore to analyze and implement critical aspects of this approach, with particular focus on the power requirements and silicon area.

This approach explores the use of a mixed analog with digital assistance architecture which uses multiple low to medium resolution ADCs with digital adaptive filters. The effective dynamic range of the front-end is enhanced by cancelling the unwanted interference in the time domain.

Interference cancellation performance is improved using oversampling and a digital dual adaptive signal processing technique that provides a low mean squared error for cancellation together with a large processing gain. The system could achieve at least 11 bit equivalent dynamic range for the desired weak signals using two 5-bit ADC's and a 7-bit DAC. In general, the Effective Bits from Dynamic Range Reduction (EBDR) for the system is equal to, or more than N+M bits, where N and M are resolutions of the two ADC's used.

The key components of the system are high speed medium resolution ADC's and they have been implemented to demonstrate the path to a low power small area solution. An asynchronous 1GS/s ADC with a peak SNDR of 31.5dB, ENOB 5.0 bits, is achieved by time interleaving two ADC's based on the binary equivalent successive approximation (SA) algorithm using a series capacitive ladder with input capacitance of 84fF. A simple extension of the SA algorithm essentially removes the ENOB degradation due to metastability from the comparator. The ADC's are fabricated in 65nm CMOS with an active area of 0.11mm², with a total power consumption of 6.7mW.

It is believed that the time domain approach for wideband, high dynamic range applications which has been explored in this thesis is a step towards the goal of a future radio system, in which the A/D conversion process is directly after the front-end low noise amplifiers.