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Ductal carcinoma in situ (DCIS) of the breast is often managed by lumpectomy and radiation or mastectomy, despite its indolent features. Effective non-invasive treatment strategies could reduce the morbidity of DCIS treatment. We have exploited the high heat shock protein 90 (HSP90) activity in premalignant and malignant breast disease to non-invasively detect and selectively ablate tumors using photodynamic therapy (PDT). PDT with the HSP90-targeting photosensitizer, HS201, can not only ablate invasive breast cancers (BCs) while sparing non-tumor tissue, but also induce antitumor immunity. We hypothesized that HS201-PDT would both non-invasively ablate DCIS and prevent progression to invasive BC. We tested in vitro selective uptake and photosensitivity of HS201 in DCIS cell lines compared to the non-selective parental verteporfin, and assessed in vivo antitumor efficacy in mammary fat pad and intraductal implantation models. Selective uptake of HS201 enabled treatment of intraductal lesions while minimizing toxicity to non-tumor tissue. The in vivo activity of HS201-PDT was also tested in female MMTV-neu mice prior to the development of spontaneous invasive BC. Mice aged 5 months were administered HS201, and their mammary glands were exposed to laser light. HS201-PDT delayed the emergence of invasive BC, significantly prolonged disease-free survival (DFS) (p = 0.0328) and tended to improve overall survival compared to the no-treatment control (p = 0.0872). Systemic administration of anti-PD-L1 was combined with HS201-PDT and was tested in a more aggressive spontaneous tumor model, HER2delta16 transgenic mice. A single PDT dose combined with anti-PD-L1 improved DFS compared to the no-treatment control, which was significantly improved with repetitive HS201-PDT given with anti-PD-L1 (p = 0.0319). In conclusion, a non-invasive, skin- and tissue-sparing PDT strategy in combination with anti-PD-L1 antibodies effectively prevented malignant progression of DCIS to invasive BC. This non-invasive treatment strategy of DCIS may be safe and effective, while providing an option to reduce the morbidity of current conventional treatment for patients with DCIS. Clinical testing of HS201 is currently underway.

Project objective: Despite the recent revolution in immune checkpoint inhibitors (ICIs), only modest improvement in overall survival and likely caused by not enough potent cellular immunity among BC patients. Our lab has been focus on inducing cellular immunity against HER2+ BC through vaccination against the tumor-associated antigen HER2. Approximately 20 years ago, we performed an experimental pilot study by administrating HER2 peptide and recombinant protein pulsed dendritic cells (DC vaccine) to six patients with refractory HER2+ advanced or metastatic (stage II (≥ 6 +LN), III, or stage IV) BC. We followed the patients on 2019 found that all of the six patients were still alive, 18 years after vaccination. Their blood sample were analyzed with cytometry by time-of-flight (CyTOF) and found there is a significantly increased presence of CD27 expressing memory T cells in response to HER2 peptide stimulation. Recent report on the SARS-CoV2 mRNA vaccine also suggested that CD27 expressing memory T cells plays a critical role in long-lasting cellular immunity against SARS-CoV2 infection. Therefore, we hypothesized that CD27 plays a critical role in cellular immunity against BC, and the stimulation of CD27 expressing T cells with mAb targeting CD27 significantly increase the cellular immunity triggered by vaccination against tumor-associated antigen. Results: We recapitulate the rise of CD27+ antigen specific T cells among the vaccinated patients using a transgenic mouse model expressing human CD27. When combined the adenoviral-vector based HER2 (Ad-HER2) vaccination with a single dose of human aCD27 antibody (Varlilumab), we found there is a robust increase in the HER2 specific T cells compared to vaccination alone, especially CD27+CD44+ memory CD4 T cells, even after 120 days post vaccination. Using an ICI-insensitive syngeneic HER2+ BC models, we found 50% of mice in the combination group of aCD27 antibody plus Ad-HER2 showed total tumor regression by the end of study. When combined with anti-PD1 antibody, the combination of AdHER2 and Varlilumab leads to total tumor regression in 90% of tumor bearing mice with syngeneic HER2+ BC, indicating that the vaccination against tumor associated antigen HER2 plus anti-CD27 antibody sensitized ICI-insensitive HER2+ BC toward ICI. Conclusions: Our data demonstrates that the administration of anti-CD27 antibody significantly increase the long term presence of CD27+ antigen specific memory T cells after vaccination against tumor associated antigen HER2. As consequence, combination of anti-CD27 with HER2 sensitized the immune unresponsive breast cancer toward anti-PD1 antibody. Our study suggests that the vaccination against tumor-associated antigen with mAb targeting CD27 leads to the robust cellular immunity, which is required for successful ICIs against breast cancer. Citation Format: Bin-Jin Hwang, Erika Crosby, Timothy Trotter, Li-Chung Tsao, Tao Wang, Xiao Yang, Herbert Kim Lyerly, Zachary Hartman. Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 696.

Background: The CD47-SIRPα axis is an innate immune checkpoint that primarily regulates myeloid cells. In the context of cancer, tumor cells upregulate CD47 to engage SIRPα inhibitory signaling to block phagocytosis. Thus, inhibition of this axis may enhance myeloid cell phagocytosis and adaptive immune responses against cancer. While our previous studies identified that CD47 mAbs enhance HER2-specific mAb therapy (Tsao et al., 2019), clinical trials have revealed that CD47-targeting mAbs are associated with severe side effects of anemia and thrombocytopenia in patients. We thus hypothesized that targeting SIRPα, which is more restrictively expressed on myeloid cells, may achieve similar but less toxic treatment effects. Methods: To ascertain the impact of SIRPα inhibition against different molecular subtypes of breast cancer, we utilized and validated a novel SIRPα KO mouse, along with SIRPα mAbs in our previously utilized models of Triple-Negative Breast Cancer (TNBC) and HER2+ Breast Cancer (Crosby et al., 2018; Tsao et al., 2019). Using both in vitro and in vivo tumor models, we tested the therapeutic impact of SIRPα inhibition/blockade combined with immunogenic chemotherapy (doxorubicin) in TNBC or combined with HER2-specific mAbs in HER2+ Breast Cancer (HER2+ BC). Results: After validating the loss of SIRPα in KO mice, we found that E0771 TNBC cells had reduced rates of engraftment in SIRPα KO mice compared to controls, but comparable rates of growth once tumors were established. We then treated E0771 engrafted SIRPα KO and wildtype mice with doxorubicin. This treatment elicited tumor rejection in the majority of SIRPα KO mice (70% of injected tumors), compared to only 10% rejection in SIRPα WT mice. To study tumor antigen-specific immunity, we expressed different forms of OVA (secreted, cytoplasmic, membrane) in tumor cells. We found that membrane expressed OVA led to universal tumor rejection (10/10 mice) with high levels of OVA-specific Abs, in contrast to non-membrane expressed forms of OVA. This suggested the importance of tumor-specific Abs in eliciting tumor rejection. To specifically test the antitumor effect of tumor-specific antibodies in the context of SIRPα blockade, we evaluated the impact of a HER2 mAb (Trastuzumab) on HER2+ BC cells. We found enhanced phagocytosis and antitumor effects by SIRPα KO macrophages in comparison to wild-type macrophages, suggesting the potential of targeting SIRPα along with the use of standard-of-care mAb. Conclusion: Our study demonstrates that SIRPα loss can prevent tumor engraftment but may not have a strong effect against established tumors without secondary immune stimulation. However, addition of immunogenic cell death-eliciting chemotherapy or tumor-specific Abs promote tumor phagocytosis and stimulate anti-tumor immunity. These results support further investigation of SIRPα-targeting combination therapies for cancer. Citation Format: Xingru Ma, Zachary Hartman, Li-Chung Tsao, Tao Wang, Cong-Xiao Liu, Xiao Yang, Gangjun Lei, Junping Wei. Inhibition of CD47-SIRPα axis to enhance immunity against different molecular subtypes of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5067.

The oncogenic property of the Src homology phosphotyrosine phosphatase 2 (SHP2) is well-known, but developing specific inhibitors has been very difficult. Based on our previous reports that showed the importance of acidic residues surrounding SHP2 substrate phosphotyrosines for specific recognition, we have rationally designed and chemically synthesized a small-molecule SHP2 inhibitor named 4,4'-(4'-carboxy)-4-nonyloxy-[1,1'-biphenyl]-3,5-diyl)dibutanoic acid (CNBDA). Molecular modeling predicted that CNBDA packs well into the SHP2 active site and makes extended interactions primarily with positively charged and polar amino acids surrounding the active site. In vitro PTPase assays showed that CNBDA inhibits SHP2 with an IC50 of 5 μM. However, the IC50 of CNBDA toward SHP1, the close structural homologue of SHP2, was 125 μM, suggesting an approximately 25-fold effectiveness against SHP2 than SHP1. Because SHP2 is known for its positive role in breast cancer (BC) cell biology, we tested the effect of SHP2 inhibition with CNBDA in HER2-positive BC cells. Treatment with CNBDA suppressed cell proliferation in 2D culture, anchorage-independent growth in soft agar, and mammosphere (tumorisphere) formation in suspension cultures in a concentration-dependent manner. Furthermore, CNBDA inhibited EGF-induced signaling and expression of HER2 by inhibiting the PTPase activity of SHP2 in BC cells. These findings suggest that CNBDA is a promising anti-SHP2 lead compound with anti-BC cell effects.

Accumulating evidence suggests that the Src homology phosphotyrosyl phosphatase 2 (SHP2) is a bona fide oncogene. These conclusions are primarily based on the positive role of SHP2 in receptor tyrosine kinase (RTK) signaling in multiple systems. Because overexpression of the human epidermal growth factor receptor 2 (HER2) is one of the primary causes of BC, we investigated the potential of SHP2 targeting in this subtype of BC. We have used our recently patented SHP2 inhibitor called WGMDY (US 9,932,288) for targeting SHP2 under cell culture and in vivo conditions. We have also used the recently reported allosteric SHP2 inhibitor called SHP099 particularly in the cell culture studies. We found that HER2-positive BC cells are highly sensitive to SHP2 inhibition with WGMDY as determined by loss of viability in 2D, transformation in soft agar, and cancer stem cell properties in suspension cultures. Similar results were obtained with SHP099 albeit at a reduced rate. Analysis of protein and RNA extracts showed inhibition of the Ras-ERK and the PI3K-Akt signaling pathways by WGMDY, but SHP099 tend to activate Akt signaling. In addition, WGMDY blocked overexpression of the HER2 protein in HER2-positive BC cells. As such, we focused on WGMDY for the in vivo efficacy studies. The results showed that WGMDY induces regression of preformed tumors in a concentration dependent manner. Histopathology analyses of tumors showed induction of necrosis in treated samples. Hence, WGMDY has a promising anti-cancer effects in HER2-positive BC. Citation Format: Yehenew Agazie, Zachary Hartman. Targeting the tyrosine phosphatase SHP2 is efficacious against HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-20.

We present high angular resolution imaging observations of 517 host stars of TESS exoplanet candidates using the `Alopeke and Zorro speckle cameras at Gemini North and South. The sample consists mainly of bright F, G, K stars at distances of less than 500 pc. Our speckle observations span angular resolutions of ~20 mas out to 1.2 arcsec, yielding spatial resolutions of, Accepted to AJ

Approximately 20% of breast cancer (BC) is caused by overexpression of the human epidermal growth factor receptor 2 (HER2). As a result, several antibody-based and small molecule-based anti-HER2 therapies have been developed. These drugs have benefited BC patients by improving overall survival and quality of life. However, development of resistance and disease recurrence have been the major clinical challenges. One way to overcome these clinical problems is to develop alternative therapeutic strategies. Multiple lines of evidence show that targeting the Src homology phosphotyrosyl phosphatase 2 (SHP2) in HER2-positive BC may prove beneficial both in treatment-naïve and anti-HER2 therapy-resistant forms of the disease. For instance, silencing SHP2 expression in HER2-positive breast cancer cells or conditional knockout in ErbB2 transgenic mice blocks HER2 overexpression and associated signaling, leading to loss of cell transformation and tumorigenesis. To test the clinical translational significance of SHP2 targeting, we have invented a specific small molecule SHP2 inhibitor named WGMDY (US 9,932,288) that has shown promising anti-cancer effects. The results obtained so far show that inhibition of SHP2 with WGMDY blocks HER2 expression, suppresses cell proliferation and anchorage independent growth, and induces regression of preformed xenograft tumors. These findings suggest that SHP2 might be an excellent drug target for HER2-positive BC and WGMDY has a promising potential to serve as a lead compound for developing anti-SHP2 drugs. Citation Format: Agazie Y, Hartman Z. Targeting SHP2 for the treatment of HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-17.

Background: The majority of colorectal carcinomas (CRCs) are insensitive to anti-PD-1/PD-L1 immune checkpoint inhibitor (ICI) antibodies. While there are many causes for ICI insensitivity, recent studies suggest that the suppression of innate immunity through a loss of innate immune adaptor expression could be a root cause of this insensitivity and an important factor in the evolution of tumor immunosuppression. Methods: We interrogated mitochondrial antiviral signaling gene (MAVS) expression through bioinformatics analyses of multiple datasets to validate its suppression in clinical samples. We then engineered MAVS expressing tumor cells and tested its ability to elicit innate and adaptive anti-tumor immunity using both in vitro and in vivo approaches, which we then confirmed using MAVS expressing viral vectors. Finally, we observed that MAVS stimulated PD-L1expression in different types of tumor cells and then assessed the combination of PD-L1 ICI antibodies with MAVS tumor expression in vivo. Results: MAVS was significantly downregulated in CRCs, but its re-expression could stimulate broad cellular interferon-related responses, in both murine and patient-derived CRCs. In vivo, local MAVS expression elicited significant anti-tumor responses in both immune-sensitive and insensitive CRC models, through the stimulation of an interferon responsive axis that elicited tumor antigen-specific adaptive immunity. Critically, we found that tumor-intrinsic MAVS expression triggered systemic adaptive immune responses that enabled abscopal CD8+ T cell cytotoxicity against distant CRCs. As MAVS also induced PD-L1 expression, we further found synergistic anti-tumor responses in combination with anti-PD-L1 ICIs. Conclusion: These data demonstrate that intratumoral MAVS expression results in local and systemic tumor antigen-specific T cell responses, which could be combined with PD-L1 ICI to permit effective anti-tumor immunotherapy in ICI resistant cancers. We are further exploring to overcome the ICI insensitivity of other types of solid tumor, with MAVS-delivery strategies other than viral vectors. Citation Format: Bin-Jin Hwang, Li-Chung Tsao, Chaitanya Acharya, Timothy Trotter, Pankaj Agarwal, Tao Wang, Junping Wei, Xiao-Yi Yang, Gang-jun Lei, Takuya Osada, Herbert Kim Lyerly, Michael A. Morse, Zachary Hartman. Sensitizing immune unresponsive colorectal cancers to immune checkpoint inhibitors through MAVS overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3531.

We present a Bayesian method to cross-match 5,827,988 high proper motion Gaia sources ($\mu>40 \ mas \ yr^{-1}$) to various photometric surveys: 2MASS, AllWISE, GALEX, RAVE, SDSS and Pan-STARRS. To efficiently associate these objects across catalogs, we develop a technique that compares the multidimensional distribution of all sources in the vicinity of each Gaia star to a reference distribution of random field stars obtained by extracting all sources in a region on the sky displaced 2$^\prime$. This offset preserves the local field stellar density and magnitude distribution allowing us to characterize the frequency of chance alignments. The resulting catalog with Bayesian probabilities $>$95% has a marginally higher match rate than current internal Gaia DR2 matches for most catalogs. However, a significant improvement is found with Pan-STARRS, where $\sim$99.8% of the sample within the Pan-STARRS footprint is recovered, as compared to a low $\sim$20.8% in Gaia DR2. Using these results, we train a Gaussian Process Regressor to calibrate two photometric metallicity relationships. For dwarfs of $3500, Comment: 51 pages, 23 figures

The Quad-camera Wavefront-sensing Six-channel Speckle Interferometer (QWSSI) is a new speckle imaging instrument available on the 4.3-m Lowell Discovery Telescope (LDT). QWSSI is built to efficiently make use of collected photons and available detector area. The instrument images on a single Electron Multiplying CCD (EMCCD) at four wavelengths in the optical (577, 658, 808, and 880nm) with 40nm bandpasses. Longward of 1µm, two imaging wavelengths in the NIR are collected at 1150 and 1570nm on two InGaAs cameras with 50nm bandpasses. All remaining non-imaging visible light is then sent into a wavefront EMCCD. All cameras are operated synchronously via concurrent triggering from a timing module. With the simultaneous wavefront sensing, QWSSI characterizes atmospheric aberrations in the wavefront for each speckle frame. This results in additional data that can be utilized during post-processing, enabling advanced techniques such as Multi-Frame Blind Deconvolution. The design philosophy was optimized for an inexpensive, rapid build; virtually all parts were commercial-off-the-shelf (COTS), and custom parts were fabricated or 3D printed on-site. QWSSI’s unique build and capabilities represent a new frontier in civilian high-resolution speckle imaging.

The burden of surgical disease in refugee and internally displaced person (IDP) populations has not been well defined. Populations fleeing conflict are mobile, limiting the effectiveness of traditional sampling methods. We employed novel sampling and survey techniques to conduct a population-based surgical needs assessment amongst IDPs in Kerenik, West Darfur, Sudan, over 4 weeks in 2008. Satellite imagery was used to identify man-made structures. Ground teams were guided by GPS to randomly selected households. A newly created surgical needs survey was administered by surgeons to household members. One randomly selected individual answered demographic and medical history questions pertaining to themselves and first-degree blood relatives. All household members were offered a physical examination looking for surgical disease. There were 780 study participants; 82% were IDPs. A history since displacement of surgical and potentially surgical conditions was reported in 38% of respondents and by 73% of respondents in first-degree blood relatives. Surgical histories included trauma (gunshots, stabbings, assaults) (5% respondents; 27% relatives), burns (6% respondents; 14% relatives), and obstetrical problems (5% female respondents; 11% relatives). 1485 individuals agreed to physical examinations. Untreated surgical and potentially surgical disease was identified in 25% of participants. We identified and characterized a high burden of surgical and potentially surgical disease in an IDP population in West Darfur. Our study is unique in its direct assessment of a traumatized, mobile, vulnerable population. Health officials and agencies charged with the care of IDP and refugee populations should be aware of the high prevalence of surgical and potentially surgical conditions in these communities. This study adds to the growing body of evidence that investment in surgical resources may address a significant portion of the overall burden of disease in marginalized populations.

We calculate directly determined values for effective temperature ($T_{\rm EFF}$) and radius ($R$) for 191 giant stars based upon high resolution angular size measurements from optical interferometry at the Palomar Testbed Interferometer. Narrow- to wide-band photometry data for the giants are used to establish bolometric fluxes and luminosities through spectral energy distribution fitting, which allow for homogeneously establishing an assessment of spectral type and dereddened $V_{\rm 0}-K_{\rm 0}$ color; these two parameters are used as calibration indices for establishing trends in $T_{\rm EFF}$ and $R$. Spectral types range from G0III to M7.75III, $V_{\rm 0}-K_{\rm 0}$ from 1.9 to 8.5. For the $V_{\rm 0}-K_{\rm 0} = \{1.9,6.5\}$ range, median $T_{\rm EFF}$ uncertainties in the fit of effective temperature versus color are found to be less than 50K; over this range, $T_{\rm EFF}$ drops from 5050K to 3225K. Linear sizes are found to be largely constant at 11 $R_\odot$ from G0III to K0III, increasing linearly with subtype to 50 $R_\odot$ at K5III, and then further increasing linearly to 150 $R_\odot$ by M8III. Three examples of the utility of this data set are presented: first, a fully empirical Hertzsprung-Russell Diagram is constructed and examined against stellar evolution models; second, values for stellar mass are inferred based on measures of $R$ and literature values for $\log g$. Finally, an improved calibration of an angular size prediction tool, based upon $V$ and $K$ values for a star, is presented., Comment: 310 pages, 16 figures, 21 tables, accepted to be published in The Astrophysical Journal

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1–4 or the human homologs HER1–4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr(992)-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

We present a catalog of 99,203 wide binary systems, initially identified as common proper motion (CPM) pairs from a subset of ~5.2 million stars with proper motions $��> 40$ mas/year, selected from Gaia data release 2 (DR2) and the SUPERBLINK high proper motion catalog. CPM pairs are found by searching for pairs of stars with angular separations $< 1$ degree and proper motion differences $����< 40$ mas/year. A Bayesian analysis is then applied in two steps. In a first pass, we use proper motion differences and angular separations to distinguish between binaries and chance alignments. In a second pass, we use parallax data from Gaia DR2 to refine our Bayesian probability estimates. We present a table of 119,390 pairs which went through the full analysis, 99,203 of which have probabilities $>95\%$ of being real binaries. Of those 99,203 high probability pairs, we estimate that only about 364 pairs are most likely to be false positives. In addition, we identify 57,506 pairs which have probabilities $>10\%$ from the first pass but have high parallax errors and therefore were not vetted in the second pass. We examine the projected physical separation distribution of our highest probability pairs and note that the distribution is a simple exponential tail and shows no evidence of being bi-modal. Among pairs with lower probability, wide binaries are detected at larger separations ($>10^{4-5}$ AU) consistent with the very wide population suggested in previous studies, however our analysis suggests that these do not represent a distinct population. We examine the HR diagram of this set of high probability wide binaries and find evidence for 980 over-luminous components among 2,227 K+K wide binaries; assuming these represent unresolved sub-systems, we determine that the higher order multiplicity fraction for K+K wide systems is at least 39.6\%., 38 Pages, 34 Figures, Accepted to ApJS, Comments are welcome

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of receptor tyrosine kinases (RTK) signaling. Furthermore, SHP2 is known to promote cell migration and invasiveness, key steps in cancer metastasis. To date, however, the mechanism by which SHP2 regulates cell movement is not fully understood. In the current report, a new role for SHP2 in regulating cell migration has been suggested. We show that SHP2 mediates lamellipodia persistence and cell polarity to promote directional cell migration in the MDA-MB231 and the MDA-MB468 basal-like and triple-negative breast cancer cell lines. We further show that SHP2 modulates the activity of focal adhesion kinase (FAK) by dephosphorylating pTyr397, the autophosphorylation site that primes FAK function. Because hyperactivation of FAK is known to counter the maturation of nascent focal complexes to focal adhesions, we propose that one of the mechanisms by which SHP2 promotes lamellipodia persistence is by downregulating FAK activity through dephosphorylation of pTyr397. The finding that inhibition of FAK activity partially restores EGF-induced lamellipodia persistence and cell migration in SHP2-silenced cells supports our proposition that SHP2 promotes growth factor–induced cell movement by acting, at least in part, on FAK. However, the effect of SHP2 inhibition in nonstimulated cells seems FAK independent as there was no significant difference between the control and the SHP2-silenced cells in pY397-FAK levels. Also, FAK inhibition did not rescue Golgi orientation defects in SHP2-silenced cells, suggesting that SHP2 acts through other mechanisms to promote cell polarity. Mol Cancer Res; 11(6); 651–64. ©2013 AACR.

One of the causes of breast cancer is overexpression of the human epidermal growth factor receptor 2 (HER2). Enhanced receptor autophosphorylation and resistance to activation-induced downregulation have been suggested as mechanisms for HER2-induced sustained signaling and cell transformation. However, the molecular mechanisms underlying these possibilities remain incompletely understood. In the current report, we present evidence that show that HER2 overexpression does not lead to receptor hyper-autophosphorylation, but alters patterns in a manner that favors receptor stability and sustained signaling. Specifically, HER2 overexpression blocks epidermal growth factor receptor (EGFR) tyrosine phosphorylation on Y1045 and Y1068, the known docking sites of c-Cbl and Grb2, respectively, whereas promoting phosphorylation on Y1173, the known docking site of the Gab adaptor proteins and phospholipase C gamma. Under these conditions, HER2 itself is phosphorylated on Y1221/1222, with no known role, and on Y1248 that corresponds to Y1173 of EGFR. Interestingly, suppressed EGFR autophosphorylation on the Grb2 and c-Cbl-binding sites correlated with receptor stability and sustained signaling, suggesting that HER2 accomplishes these tasks by altering autophosphorylation patterns. In conformity with these findings, mutation of the Grb2-binding site on EGFR (Y1068F-EGFR) conferred resistance to ligand-induced degradation, which in turn induced sustained signaling, and increased cell proliferation and transformation. These findings suggest that the Grb2-binding site on EGFR is redundant for signaling, but critical for receptor regulation. On the other hand, mutation of the putative Grb2-binding site in HER2 (Y1139) did not affect stability, signaling or transformation, suggesting that Y1139 in HER2 may not serve as a Grb2-binding site. In agreement with the role of EGFR in HER2 signaling, inhibition of EGFR expression reduced HER2-induced anchorage-independent growth and tumorigenesis. These results imply that complementing HER2-targeted therapies with anti-EGFR drugs may be beneficial in HER2-positive breast cancer.

Helper T cells respond to peptide antigens derived from exogenous sources presented by MHC II on antigen presenting cells. Antigens from pathogens are internalized by professional antigen presenting cells (APC) and processed for presentation. Certain epitopes are selected during processing as the final peptides for stimulation of T cells and are termed "immunodominant". Understanding how selection of immunodominant epitopes takes place has been a difficult task because of the complexity of the mechanisms governing both antigen processing and T cell recognition. In this review, we discuss our current understanding of HLA-DM function in peptide exchange and selection and its relevance to epitope immunodominance.

During this research period, molecular modeling and biochemical studies were conducted to assess the roles of different acidic residues in the binding of the Src homology 2-containing protein tyrosine phosphatase (SHP2) to its substrates. A peptide derived from this substrate was shown to be a selective inhibitor of SHP2. SHP1 was used as a control, and the peptide did not act on this enzyme. Molecular modeling revealed key binding determinants at the atomic level present in SHP2 that could not be recapitulated in SHP1. These modeling observations were tested using mutation of the peptide. Indeed, when the peptide was mutated to abolish these interactions, inhibitory capacity was abolished entirely. Systematic mutation of the peptide in silico further demonstrated that the substrate needed to bind both of the positively-charged amino acid sidechains adjacent to the active site to facilitate the highest binding stability. Mutation of any of the acidic amino acids next to the phosphotyrosine on the substrate produced significant defects in binding as predicted by molecular docking. These findings are now being parlayed into study of the peptide-based inhibitor on cells, which is work that will continue into the coming year.

For the past two decades, adenoviral vector platforms have been used as both gene therapy vehicles and vaccine platforms in various human clinical trials. However, the use of these, as well as other viral vector platforms, has been tempered by the innate immune and adaptive immune responses elicited. Despite the development of advanced generation adenoviral vectors, the innate responses precipitated by these vectors have remained largely unchanged due to the particular nature of the viral capsid and infectious process. To more effectively use these vectors for different purposes, we constructed adenoviral vectors encoding overexpression cassettes for various TLR adaptors (MyD88 and TRIF), RIG-I pathway adaptor (MAVS), as well as immuno-modulatory viral genes (pp65) whose expression might accentuate or inhibit the innate immune response enabled by adenoviral infection. Our studies show that while all of these immuno-modulatory vectors were able to effectively enhance (TLR and MAVS vectors) or inhibit (pp65 vectors) the NF-kB and IFN-beta responses in vitro, only certain vectors were able to affect the adaptive responses elicited in vivo. While our findings show that vector manipulation can influence vector-specific innate and adaptive immune responses, they also offer evidence for the highly regulatory nature of the innate response, as evidenced by the limited impact of certain adaptor overexpression.

Background: Of all breast cancer subtypes, the basal-type/triple-negative breast cancer (BTBC) has the worst clinical outcome. The major reasons are the highly invasive nature of the disease and the limited treatment options available to patients. As opposed to the HER2−positive and the hormone-positive breast cancers, targeted therapy against BTBC is unavailable. These premises underscore the need for discovering and characterizing drug targets in BTBC. In this study, we have focused on the Src homology phosphotyrosyl phosphatase 2 (SHP) which seems to play pivotal roles in BTBC. The major reason for focusing on SHP2 is that it is an essential downstream effector of mitogenic and cell survival signaling downstream of receptor tyrosine kinases such as EGFR (HER1) and IGF-1R, and the cytoplasmic tyrosine kinase Src, which are known to be elevated in BTBC. Materials and Methods: The state of SHP2 and EGFR expression in BTBC tumors was determined by immunohistochemistry. The functional significance of SHP2 in BTBC cells was investigated by ablating its expression with specific shRNA and then assessing impact on mitogenic and cell survival signaling using immunoblotting with phospho-specific antibodies, effect on transformation using 3D cultures such as growth in soft agar and matrigel, consequence on cell motility and invasiveness using florescent-tagged matrigel. Furthermore, the impact of SHP2 inhibition on tumorigenesis was tested by intramammary transplantation in vivo and monitoring and analyzing tumor growth and metastasis. Results: We have shown that the SHP2 protein is elevated in BTBC tumors. More importantly, the elevated expression of SHP2 is highly correlated with overexpression of the EGFR (HER1), suggesting their potential synergistic role to promote BTBC. Inhibition of SHP2 in BTBC cells reversed transformation and suppressed proliferation, indicating their dependence on the function of SHP2. Moreover, inhibition of SHP2 abolished EGF-induced mitogenic and cell survival signaling, which is in agreement with its role in cell proliferation and transformation. More dramatic was that the invasive phenotype of BTBC cells in 3D matrigel was completely blocked by inhibition of SHP2, suggesting that the invasive property of these cells is dependent on SHP2. Even more dramatic was that the tumorigenic and metastatic potential of BTBC cells was abolished by SHP2 inhibition. Discussion: The current study demonstrates that SHP2 plays a pivotal role in promoting BTBC. Given that SHP2 is a tyrosine phosphatase with positive signaling role, its promotion of BTBC must occurs through promotion of tyrosine kinase signaling. Together, our results provide the first glimpse on the potential of SHP2 as a drug target in BTBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-15.

Background: The Src homology phosphotyrosyl phosphatase 2 (SHP2) acts as a transducer of mitogenic and cell survival signaling downstream of receptor tyrosine kinases, cytokine receptors and integrins. As such, SHP2 promotes cell growth, transformation, survival, and motility, among other processes. Recent studies suggest that SHP2 is overexpressed in breast cancer and is essential for the maintenance of the transformed phenotype both in the HER2−positive and the basal-type/triple-negative breast cancer (BTBC) cells. Materials and Methods: The expression of SHP2 in BTBC cells was suppressed constitutively by lenti virus-mediated transduction of specific shRNA. Impact of SHP2 inhibition on cell migration was determined by the monolayer wound healing assay. The SHP2 substrate-trapping mutant was used to identify FAK as an SHP2 substrate. Site-directed mutagenesis, binding studies and phosphatase assays were employed to further characterize FAK as a bona fide SHP2 substrate. Immunofluorescence microscopy was conducted to visualize the amounts of phosphorylated FAK and its location in the cell after adhesion. Results: We demonstrate that SHP2 is essential for the migration of BTBC cells as evidenced by loss of the enhanced migratory behavior upon its inhibition. Using the SHP2 substrate-trapping mutant, FAK was identified as a biological substrate of SHP2. Site-directed mutagenesis, binding studies and phosphatase assays confirmed that SHP2 indeed associates with and dephosphorylates FAK at the pY397 site, a site known to promote the focal adhesion activity of FAK. Immunofluorescence of breast cancer cells with SHP2 inhibition were found to have higher levels of FAK pY397 further confirming that FAK is an SHP2 substrate. The increase in pY397 level correlated with loss of cell polarity, enhanced focal adhesion formation, and acquisition of non-transformed morphology. Discussion: These results suggest that dephosphorylation of pY397 of FAK by SHP2 is the major mechanism by which it is able to modulate cancer cell migration. Together with previous findings, the current studies show that SHP2 is a suitable target for therapeutic intervention to combat breast cancer metastasis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-05.

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in receptor tyrosine kinase-induced mitogenic and cell survival signaling. In addition, SHP2 mediates cell transformation induced by oncogenic tyrosine kinases. Here, we provide evidence that show that the SHP2 protein is overexpressed in basal-type/triple-negative breast cancer (BTBC) with strong correlation to higher tumor grade, metastasis and elevated expression of the epidermal growth factor. We further show that SHP2 plays an essential role in the tumorigenesis and metastasis of BTBC cells (MDA-MB-231 and MDA-MB-468) transplanted into the mammary glands of NOD/SCID mice. These results suggest that SHP2 can be used for targeted therapy in BTBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 238. doi:10.1158/1538-7445.AM2011-238

Human Adenovirus Type 4 is widely known as the responsible agent for epidemic outbreaks of Acute Respiratory Disease in military recruits. Although studied for over 50 years, we still have precious little insight into the epidemiology and high viral morbidity surrounding HAdV-4 infections. To address these issues we developed an attenuated HAdV-4 vector system as a basic tool for viral and immunologic research. Our plasmid-based system allows for the manipulation of the HAdV-4 genome, which we used to produce replication attenuated [E1−,E3−] LacZ+ 1st generation Ad4 vectors. Using these vectors, we found that Ad4 elicits an enhanced immunogenic response (comprised of significantly greater IL-6, MCP-1, RANTES, and G-CSF) in murine macrophages in vitro and upon intravenous injection in C57/BL6 mice in vivo. The enhanced immunogenicity is capsid-dependant and is partially complement-dependent in vivo for certain cytokines, related to a more pronounced thrombocytopenia upon i.v. administration. Surprisingly, our studies demonstrate that the HAdV-4 early immunogenicity correlates with suppressed IgG responses, despite different routes of administration for the vector encoded LacZ transgene. These results demonstrate that capsid specific recognition is of paramount importance to both innate and adaptive immune responses and should be an important consideration in vaccine development.

While HER2/neu specific antibody (Herceptin) therapy has clear efficacy against HER2 overexpressing tumors, most patients who initially respond to Herceptin show disease progression within one year despite continued high expression of HER2. As an alternative to Herceptin, we developed a novel set of adenoviral vectors encoding human HER2 which break tolerance and induce T and B cell responses in human-HER2-transgenic (HuHER2-Tg) mice (kindly provided by W-Z. Wei, Karmanos Cancer Center, MI). Ad-HER2 vaccination results in a high level of HER2-specific T cells and anti-HER2 vaccine induced antibodies (VIA), which significantly inhibits the growth of human HER2 expressing murine 4T1 breast cancer cells (kindly provided by M. Kershaw, Peter MacCallum Cancer Centre, Melbourne, Australia) in HuHER2-Tg mice. Anti-HER2 VIA strongly bound to Herceptin-refractory human breast tumor cells and significantly inhibited their growth in vitro, and mediated complement dependent cytotoxicity. Thus, our findings demonstrate that Ad-HER2 vaccination proved to be efficacious and therapeutically relevant in Herceptin refractory tumor models. Grant support: NIH P50-CA068438 SPORE in Breast Cancer & DOD CTRA BC050221.