Kevin Zhang

MicroRNA93 regulates proliferation and differentiation of normal and malignant breast stem cells

MicroRNAs (miRNAs) play important roles in normal cellular differentiation and oncogenesis. microRNA93 (mir-93), a member of the mir106b-25 cluster, located in intron 13 of the MCM7 gene, although frequently overexpressed in human malignancies may also function as a tumor suppressor gene. Using a series of breast cancer cell lines representing different stages of differentiation and mouse xenograft models, we demonstrate that mir-93 modulates the fate of breast cancer stem cells (BCSCs) by regulating their proliferation and differentiation states. In “claudinlow” SUM159 cells, expression of mir-93 induces Mesenchymal-Epithelial Transition (MET) associated with downregulation of TGFβ signaling and downregulates multiple stem cell regulatory genes, including JAK1, STAT3, AKT3, SOX4, EZH1, and HMGA2, resulting in cancer stem cell (CSC) depletion. Enforced expression of mir-93 completely blocks tumor development in mammary fat pads and development of metastases following intracardiac injection in mouse xenografts. The effect of mir-93 on the CSC population is dependent on the cellular differentiation state, with mir-93 expression increasing the CSC population in MCF7 cells that display a more differentiated “luminal” phenotype. mir-93 also regulates the proliferation and differentiation of normal breast stem cells isolated from reduction mammoplasties. These studies demonstrate that miRNAs can regulate the states and fates of normal and malignant mammary stem cells, findings which have important biological and clinical implications.

Targeted Oncolytic Herpes Simplex Viruses for Aggressive Cancers

Herpes simplex virus (HSV) is a well-known vector that is often used for gene therapy to treat cancers. The most attractive feature of HSV is its ability to destroy tumors through a distinctive oncolytic mechanism where the virus can destroy cancer cells via cell lysis, a killing function that no anti-cancer drugs can mimic. Importantly, HSV is a safe and effective virus that can be easily manipulated to preferentially replicate in tumor cells. In the last 20 years of reengineering efforts, a number of HSV designs, including the classical G207, have been focused on deleting viral genes in order to render the virus tumor specific. Although such designs can successfully destroy tumor xenografts in animal models, with minimal impact on normal tissues, a common trade-off is the marked attenuation of the virus. This problem is most profound in many clinical tumors, where virus dissemination is often hindered by the difficult cellular and molecular terrain of the human tumor mass. In order to harness all of HSVs replication potential to destroy tumor cells, efforts in our lab, as well as others, last several years have been focused on engineering an oncolytic HSV to target tumor cells without deleting any viral genes, and have since generated highly tumor specific viruses including our transcriptional translational dually regulated HSV (TTDR-HSV). In this review, we will discuss the improvements associated with the newer TTDR-HSV design compared to the classical defective HSV designs such as G207 and tk- HSV. Lastly, we will review additional cellular features of aggressive tumors, such as their immense cellular heterogeneity and volatility, which may serve to hinder the dissemintation of TTDR-HSV. The challenge for future studies would be to explore how TTDRHSV could be redesigned and/or employed with combinatorial approaches to better target and destroy the heterogeneous and dynamic cell populations in the aggressive tumor mass.

Abstract LB-279: Quantitative flux measurements of serum protein catabolism in PDAC and other KRAS-mutant cancers

Activating KRAS mutations are prevalent in human cancer and are associated with poor prognosis. Among these malignancies is pancreatic ductal adenocarcinoma (PDAC), which is universally KRAS-driven and almost universally lethal. PDAC tumors are extremely fibrotic and hypovascularized, limiting perfusion of nutrients into the tumor, and tumor cells exhibit an altered metabolic program to support survival and proliferation in this environment. One metabolic activity upregulated in these tumor cells is the uptake and catabolism of serum protein. This process yields a substantial alternative source of amino acids and can support the proliferation of cultured cells lacking free essential amino acids. However, existing methods for assaying the uptake and degradation of intact protein provide qualitative outputs, and as such, do not yield accurate estimates of flux from serum protein to amino acid monomers. Here, we present a method for quantitative measurement of the catabolic production of amino acids from serum protein in cultured cells. By culturing cells in medium containing fully 13C-labeled glucose and amino acids supplemented with unlabeled albumin, we distinguish amino acids taken up as monomers from the medium from serum protein-derived amino acids. Using a variant of classical metabolic flux analysis, we derive flux estimates for serum protein catabolism from measurements of amino acid abundance and isotopic labeling over time. Our method is highly sensitive (i.e. can estimate “baseline” serum protein catabolism in KRAS wild-type cell lines) and yields precise estimates of amino acid influx from serum protein for all proteinogenic amino acids. We have measured serum protein catabolism in a variety of cultured cell lines and find that protein catabolism yields amino acids in comparable amounts to conventional uptake in various pancreatic and non-pancreatic lines. This approach enables estimation of protein catabolic flux in any cell line and can be used to assay the effects of various genetic and pharmacological perturbations on serum protein catabolism with high sensitivity and while accounting for growth rate differences. Citation Format: Michel Nofal, Kevin Zhang, Josh Rabinowitz. Quantitative flux measurements of serum protein catabolism in PDAC and other KRAS-mutant cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-279. doi:10.1158/1538-7445.AM2015-LB-279

Abstract LB-60: Targeting stem cells in triple negative breast cancer through combined MEK and AKT inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Breast cancer stem cells (CSCs) are hypothesized to be involved in tumor metastasis, dormancy, and eventual recurrence. Among the molecular subtypes of breast cancer, triple negative (i.e. lacking HER2 amplification and estrogen and progesterone receptors) cancers have the highest frequency of CSCs with cytotoxic chemotherapy being the only established treatment option. Breast CSCs are relatively resistant to cytotoxic chemotherapy and radiation treatment indicating new treatment modalities are needed. Breast CSCs can be identified using flow cytometry with the cell surface marker profile CD44+CD24-EpCAM+ or by intracellular expression of aldehyde dehydrogenase (ALDH+). Previously, we identified a role for AKT signaling in the survival of breast CSCs. Here, we have tested if inhibition of MEK and AKT signaling with two small molecules, GSK1120212 and GSK690693, respectively, were able to reduce the frequency and overall number of CD44+CD24-EpCAM+ and/or ALDH+ CSCs in multiple triple-negative breast cancer cell lines. Our results suggest that either the CD44+CD24-EpCAM+ or ALDH+ fraction are sensitive to three days of combined inhibitor treatment. Within the MDA-MB-468 cell line, there is more phospho AKT (S473) and phospho MEK1/2 (S218/S222) signaling in both ALDH+ and CD44+CD24-EpCAM+ cells compared to non-stem cells. Furthermore, there is more phospho MEK1/2 in ALDH+ cells compared to CD44+CD24-EpCAM+ and more phospho AKT in CD44+CD24-EpCAM+ cells compared to ALDH+ CSCs. Together, these results highlight the possibility of using a combination of these agents in clinical trials to target triple-negative breast CSCs. Citation Format: Kevin Zhang, Sean P. McDermott, Max S. Wicha. Targeting stem cells in triple negative breast cancer through combined MEK and AKT inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-60. doi:10.1158/1538-7445.AM2014-LB-60

Abstract 5022: Early progenitor cells but not late luminal progenitor cells preserve in invasive breast carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background CD133 and CD117 represent markers of human breast tissue differentiation. CD133 is an early progenitor marker while CD117 represents late luminal progenitor marker (Lim, et al, Nature Medicine 2009). Morphologic expression of the two markers has not been well established in paraffin embedded tissue of human breast lesions. Design In this study, we evaluated CD133 and C117 expression in different human breast lesions. Group 1 consisted of 15 benign breast cases (5 reduction mammoplasty cases with no significant pathology and 10 fibroadenoma or fibrocystic changes). Group 2 (malignant group 2) included 13 breast cases with invasive ductal carcinoma (IDC). Group 3 (malignant group 3) was composed of 12 BRCA-mutant breast cases but only six cases had IDC. All sections from paraffin embedded tissue were immunohistochemically stained for CD133 (AC133 clone) and CD117 (monoclonal antibody). Expression of the two markers in normal breast glands adjacent to lesions (normal controls), benign lesions (benign controls) and IDC were evaluated and positive rate was calculated. Results Both CD133 and CD117 were expressed in normal breast glands (normal controls) of all three groups and benign breast lesions ([Table 1][1]). However, the expression of CD133 was detected in the majority of IDC lesions as compared to low positive percent of CD117 expression in both groups 2 and 3 ([Table 1][1]). Recombined tumor cases, based on the molecular luminal status, also showed 56 - 70% CD133 positivity but 0 - 20% CD117 positivity in IDC cells. View this table: Table 1. CD133 and CD117 in benign and malignant breast lesions Conclusions We show that normal breast glands expressed both CD133 and CD117, compatible with the presence of stem cell niche in normal breast ducts and acini. However our data suggest that in IDC, there is presence of early progenitor cells but not late luminal progenitor cells, regardless of the molecular luminal status, supporting the view that cancer stem cells exist in the invasive breast carcinoma. Citation Format: Kevin J. Zhang, Suling Liu, Nava Siegelmann-Danieli, Ping L. Zhang. Early progenitor cells but not late luminal progenitor cells preserve in invasive breast carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2013-5022 [1]: #T1

Abstract 3310: microRNAs regulate the transition between EMT and MET breast cancer stem cell states

Recent studies have suggested similarities between cancer stem cells and the epithelial mesenchymal transition (EMT) state. In contrast, other studies suggest that these “states” are mutually exclusive. Our studies suggest that these divergent views may be explained by the existence of multiple stem cell states, which are regulated by microRNAs. Utilizing primary breast tissue and established cell lines, we demonstrate that both normal and malignant breast stem cells exist in distinct, inter-convertible states. The EMT-like state is characterized by expression of vimentin and N-cadherin, slug, snail and twist transcription factors. EMT-like CSCs have a mesenchymal morphology, are largely quiescent, invasive and characterized by expression of the CSC markers CD44+CD24− and are EpCAM−CD49f+. In contrast, the MET (mesenchymal epithelial transition) state of CSCs is characterized by an epithelial morphology and expression of E-cadherin and EpCAM. MET-like CSCs undergo self-renewal and express the CSC marker Aldehyde dehydrogenase (ALDH) and are EpCAM+CD49F+. A subpopulation of cells expressing both CD44+CD24− and ALDH may represent cells in transition between these states. This transition is regulated by signals in the microenvironment which in turn modulate microRNA networks. Expression induction of mir100 in MCF10A cells and several cancer cell lines resulted in a decrease of ALDH-positive CSC population with a concomitant increase in the CD24−CD44+ population accompanied by induction of EMT. We demonstrated mir100 effects are mediated by targeting BMPR2, SMARCA5 and SMARCD1, all of which may contribute to induction of EMT. Moreover, we show that mir100 overexpression induces cellular quiescence as shown by Ki67 and Brdu staining. Induction of mir100 expression immediately upon orthotopic implantation or after the tumors are established significantly reduced the subsequent tumor growth in NOD/SCID mice. In contrast of overexpression of mir100 stimulates cell invasion in vitro matrigel assay. The existence of multiple cancer stem cell states has important implications for understanding stem cell plasticity as well as tumor growth and metastasis. In addition, the existence of these states has implications for the development of CSC-targeting therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3310. doi:1538-7445.AM2012-3310

Abstract 4167: Establishment and characterization of a nude mouse model directly derived from human primary colon cancer

The human tumor models established by inoculation of transformed tumorigenic tumor cell lines in immunodeficient mice have long been used in drug RD however, the prediction for clinical effectiveness is not satisfactory by far. One of the obvious gaps is due to the adaptation of the tumor lines in culture environment and/or in vivo passages that make them step away from the original clinical tumor, e.g. in the reproducibility of the development and malignant behaviors, the specific gene expression profiles and the responsiveness to anti-cancer drug treatment, etc. We took a strategy of directly inoculation of the tumor tissues collected from Chinese colon cancer patients to establish human primary colon cancer model in nude mice. We successfully kept the tumor survived in vivo, but within limited passages to minimize the unnecessary adaptive change. One of the primary colon cancer models established with subcutaneous inoculation (HPTCSC-10-3) was further characterized. The histopathological analysis of the tumors from each passage showed the characters of a moderately differentiated colon epithelial adenocarcinoma with gland-like columnar epithelial lining mixed with mucin secretion, identical to the original tumor. The tumor growth rate following subcutaneous xenograft in nude mice was sensitive to the treatment of multiple drugs: 69%, 50% and 77% inhibition in response to the treatment for 3 weeks with CPT-11 (10 mg/kg, i.v., twice weekly), Docetaxel (2 mg/kg, i.v., twice weekly) and Sutent (40 mg/kg, p.o., daily), respectively. A 90.1% inhibition of tumor growth was reached by CPT-11 treatment at same dosing regimen following orthotopic xenograft in cecum. The primary cells of HPTCSC-10-3 tumor demonstrated to be more sensitive to Dox treatment compared to HCT-116 (IC50: 456 vs. 650 ng/ml) in MTT assay. Gene analysis of the tumor tissues using limited panel RT-PCR revealed that the expression of quit a few genes, such as Erk-1 and MMP-2, were increased in a passage-dependent pattern, indicating the adaptation activities following in vivo passage. The data demonstrated that a novel human colon cancer model was successfully established. The preliminary characterization indicated that this model was highly representative of a clinical colon cancer when used within 7 passages, providing a useful set of tools for investigation of anticancer intervention by subcutaneous or othortopic xenograft in nude mice, or assays in the cell culture. The collection of such human primary tumor models will greatly facilitate the translational studies of anti-cancer compounds in Chinese colon cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4167.