Xiang Gu

Attenuation of TGF-β signaling supports tumor progression of a mesenchymal-like mammary tumor cell line in a syngeneic murine model.

Previous studies have suggested that TGF-β functions as a tumor promoter in metastatic, mesenchymal-like breast cancer cells and that TGF-β inhibitors can effectively abrogate tumor progression in several of these models. Here we report a novel observation with the use of genetic and pharmacological approaches, and murine mammary cell injection models in both syngeneic and immune compromised mice. We found that TGF-β receptor II (TβRII) knockdown in the MMTV-PyMT derived Py8119, a mesenchymal-like murine mammary tumor cell line, resulted in increased orthotopic tumor growth potential in a syngeneic background and a similar trend in an immune compromised background. Systemic treatment with a small-molecule TGF-β receptor I kinase inhibitor induced a trend towards increased metastatic colonization of distant organs following intracardiac inoculation of Py8119 cells, with little effect on the colonization of luminal-like Py230 cells, also derived from MMTV-PyMT tumors. Taken together, our data suggest that the attenuation of TGF-β signaling in mesenchymal-like mammary tumors does not necessarily inhibit their malignant potential, and anti-TGF-β therapeutic intervention requires greater precision in identifying molecular markers in tumors with an indication of functional TGF-β signaling.

Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERβ) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

A Novel TGFβ Trap Blocks Chemotherapeutics-Induced TGFβ1 Signaling and Enhances Their Anticancer Activity in Gynecologic Cancers

Purpose: We investigated the mechanisms of how TGFβ pathway is activated by chemotherapeutics and whether a novel TGFβ trap called RER can block chemotherapeutics-induced TGFβ pathway activation and enhance their antitumor activity in gynecologic cancer. Patients and Methods: An unbiased bioinformatic analysis of differentially expressed genes in 31 ovarian cases due to chemotherapy was used to identify altered master regulators. Phosphorylated Smad2 was determined in 30 paired cervical cancer using IHC. Furthermore, the effects of chemotherapeutics on TGFβ signaling and function, and the effects of RER on chemotherapy-induced TGFβ signaling were determined in gynecologic cancer cells. Results: Chemotherapy-induced transcriptome alteration in ovarian cancer was significantly associated with TGFβ signaling activation. Chemotherapy was found to activate TGFβ signaling as indicated by phosphorylated Smad2 in paired cervical tumor samples (pre- and post-chemotherapy). Similar to TGFβ1, chemotherapeutics were found to stimulate Smad2/3 phosphorylation, cell migration, and markers related to epithelial–mesenchymal transition (EMT) and cancer stem cells (CSC). These TGFβ-like effects were due to the stimulation of TGFβ1 expression and secretion, and could all be abrogated by TGFβ inhibitors including a novel TGFβ trap protein called RER both in vitro and in vivo. Importantly, combination treatment with RER and cisplatin showed a higher tumor inhibitory activity than either agent alone in a xenograft model of ovarian cancer. Conclusions: Chemotherapeutics can stimulate TGFβ1 production and consequently enhance TGFβ signaling, EMT, and CSC features resulting in reduced chemo-sensitivity. Combination therapy with a TGFβ inhibitor should alleviate this unintended side effect of chemotherapeutics and enhance their therapeutic efficacy. Clin Cancer Res; 24(12); 2780–93. ©2018 AACR.

Aging is associated with an expansion of CD49f hi mammary stem cells that show a decline in function and increased transformation potential

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands.

Differential effects of GLI2 and GLI3 in regulating cervical cancer malignancy in vitro and in vivo

Advanced, recurrent, or persistent cervical cancer is often incurable. Therefore, in-depth insights into the molecular mechanisms are needed for the development of novel therapeutic targets and the improvement of current therapeutic strategies. In this study, we investigated the role of GLI2 and GLI3 in the regulation of the malignant properties of cervical cancer. We showed that down-regulation of GLI2, but not GLI3, with an inducible GLI2 shRNA inhibited the growth and migration of cervical cancer cell lines, which could be rescued by ectopic expression of GLI2. GLI2 appeared to support cell growth by regulating the mitosis, but not the apoptosis, of the cervical cancer cells. Mechanistically, these functions of GLI2 were in part mediated by the activation of AKT pathway. Knockdown of GLI2, but not GLI3, also inhibited xenograft growth of cervical cancer cells in vivo. Finally, analysis of TCGA data showed that high levels of GLI2, but not GLI3, conferred a poor prognosis in cervical cancer patients. These observations for the first time suggest that GLI2, but not GLI3, exerts a tumor-promoting role in cervical cancer and may be targeted as a novel therapeutic strategy.GLI transcription factors, which mediate the Hedgehog pathway, are over-expressed in cervical cancer. RNA interference reveals an essential role of GLI2, but not GLI3, in promoting proliferation and migration and xenograft tumor growth of human cervical cancer cells. Mechanistically, the function of GLI2 is mediated by protein kinase B (AKT), and may be targeted as a novel therapeutic strategy.

Abstract 2424: Hepatocellular carcinoma in the South Texas Latino population: Implications of STEAP2

Introduction: Hepatocellular carcinoma (HCC) is the most common type of liver cancer in adults and the third most common cause of cancer death worldwide; while incidence and mortality rates are two times higher in Latinos, incidence rates are the highest among Latinos in the South Texas region. The genetic and epigenetic events associated with the increased incidence of HCC in this population are largely unknown. We performed whole genome RNA sequencing in paired HCC tumor and adjacent non-tumor tissue total RNA from nine South Texas Latino patients. Analysis of differentially expressed genes revealed significant alterations in pathways associated with oxidative stress; most importantly, we found that the expression of STEAP2 (Six Transmembrane Epithelial Antigen of the Prostate 2) is increased five-fold in HCC tumor tissue compared to adjacent non-tumor tissue. In comparison to a non-Latino population, this finding was unique to South Texas Latinos. STEAP2 is a metalloreductase of iron and copper; reduced iron and copper ions can mediate the production of hydroxyl radicals resulting in increased oxidative stress, which can cause DNA damage and lipid peroxidation. We aim to prove that STEAP2 through regulation of iron and copper homeostasis, and an increase in oxidative stress, will lead to malignant transformation of hepatocytes resulting in tumor progression of HCC, including in obese hosts. Material and Methods: Latino paired HCC and adjacent non-tumor tissues were collected for RNA sequencing, metal ion measurement and oxidative stress markers. STEAP2 RNA and protein expression levels in Latino and Caucasian samples were evaluated by RT-PCR, Western blot, and immunohistochemistry. HCC cell lines (SNU398 and HUH7) with knockdown (KD) and overexpression (OE) of STEAP2 were created to examine the proliferation, migration, anchorage independent growth, and oxidative stress in vitro. Results: Analysis of RNA sequencing data demonstrated the overexpression of STEAP2 in HCC tumors in Latino patients, which were validated by RT-PCR and Western blot data. Lipid peroxidation product, 4-hydroxynonenal, and copper levels were higher in HCC tumor vs. adjacent tissue. KD of STEAP2 in the HCC cell lines decreased proliferation, migration and anchorage independent growth, while OE of STEAP2 increase migration and anchorage independent growth but not proliferation. Conclusions: STEAP2 is specifically overexpressed in HCC tumors in Latinos in comparison to HCC tumors in non-Latino whites and appears to play a malignant-promoting role in HCC cells. Further studies on the role of STEAP2 as a novel tumor promoter in HCC and the mechanisms by which it promotes carcinogenesis are underway. The proposed studies will likely yield mechanistic insights into the molecular mechanisms that drive HCC development and progression in South Texas Latinos and potential therapeutic targets. Citation Format: Carla Zeballos, Hakim Bouamar, Guixi Zheng, Xiang Gu, Yidong Chen, Francisco G. Cigarroa, Lu-Zhe Sun. Hepatocellular carcinoma in the South Texas Latino population: Implications of STEAP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2424.

Abstract 914: Age-associated neoplastic transformation of mammary stem cells

Incidence rate of breast cancer increases significantly with age. However, the mechanism of age-associated mammary tumorigenesis is unknown. Although mammary stem/progenitor cells have been proposed as the cells of origin for breast cancer, the mechanism remains largely elusive. Previously we discovered that in the old (28 to 30 month-old) mouse mammary gland, the luminal cells in the hyperplastic lesions elevated expression of CD49f, which is a basal cell marker. This led to an increased percentage of mammary stem cells (MaSCs) marked by CD49fhi in the old in comparison to the young (3 to 6 month-old). Similarly, old (>60 yr) human mammary ducts in the adjacent normal tissues from patients with breast cancer showed more hyperplastic lesions and CD49fhi luminal cells than young ( Citation Format: Xiang Gu, Hakim Bouamar, Kyle Pressley, LuZhe Sun. Age-associated neoplastic transformation of mammary stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 914. doi:10.1158/1538-7445.AM2017-914

Abstract 4127: Blockade of TGFβ signaling enhances anticancer effect of chemotherapeutics in gynecologic cancers

Chemoresistance is a significant clinical challenge in the management of gynecologic malignancies. Previous studies have showed that TGF-β signaling pathway can mediate chemotherapy resistance. However, the underlying mechanisms of how TGF-β pathway is activated and causes chemoresistance are not well understood. Here, we found that ovarian cancer patients displayed an increased RNA transcripts of genes associated with TGF-β signaling after chemotherapy. In vitro, treatment with four commonly used drugs for gynecologic malignancies, including cisplatin, doxorubicin, paclitaxel and camptothecin, activated TGFβ signaling by stimulating phosphorylation of Smad2 and Smad3, two intracellular mediators of TGFβ signaling pathway in ovarian and cervical cancer cell lines. Furthermore, cisplatin and doxorubicin can induced epithelial-mesenchymal transition (EMT) and promote migration. Blockade of TGFβ signaling abrogated chemotherapeutics-stimulated Smad phosphorylation, epithelial-mesenchymal transition, and migration. These observations suggest that chemotherapy-mediated activation of TGFβ signaling may be a mechanism of chemoresistance, and that TGFβ pathway inhibitor may prevent the development of chemoresistant. We are currently testing the anti-tumor efficacy of the combination therapy with a novel pan-TGFβ inhibitor and cisplatin in a mouse xenograft model of ovarian cancer and whether the treatment with TGFβ inhibitor can alleviate symptoms of nephrotoxicity, which is known to be caused by cisplatin. Our studies may reveal new strategies for the treatment of gynecologic malignancies. Citation Format: Haiyan Zhu, Junhua Yang, Xiang Gu, Luzhe Sun. Blockade of TGFβ signaling enhances anticancer effect of chemotherapeutics in gynecologic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4127.

Abstract 1506: Identification of genes with age-dependent expressions in breast cancer patients

Age is the number one risk factor for breast cancer development. The breast cancer incidence rate increases with age, following beta distribution, which is approximately linear in range from 30 to 70 years old [1]. Transcriptome alterations have been shown to promote tumorigenesis for many types of cancers. Therefore, we hypothesize that the genes with altered expression during aging may promote breast cancer development. Using TCGA data, we extracted whole transcriptome profiling data of matched normal tissues from 82 female patients with age at diagnosis and menopausal status available. The RSEM estimated raw reads counts data were normalized by library sizes, log2 transformed and further normalized by quantile normalization. Removal of 25% of genes with lowest mean expressions resulted in subsequent analyses on 15,398 genes. We applied simple linear regression to study the association between gene expression level and age at diagnosis on all the 82 patients, and on the 54 post-menopausal patients to reduce the leverage effect of pre-menopausal patients with limited age range. The genes with significant correlation are filtered by the criteria: 1) R2 value is among the highest 5%, 2) absolute value of slope for age is among the highest 5% and 3) adjusted p value for the slope is among the lowest 5%. We identified 210 upregulated and 98 downregulated genes in all patients, as well as 103 upregulated and 164 downregulated genes in post-menopausal patients only. The unions of these genes (258 upregulated and 240 downregulated) are considered as genes affected by age. A combination of methods using moderate hierarchical t test (R/limma) and moderate fitting based on negative binomial distribution (R/DESeq2), with and without Surrogate Variable Analysis for potential confounders (R/SVA), results in identification of 498 upregulated and 256 downregulated genes altered by menopause by comparing post-menopausal to pre-menopausal patients (FDR Funding: CPRIT Research Training Award (RP140105) Reference: 1. Francesco Pompei and Richard Wilson (2001). Age Distribution of Cancer: The incidence Turnover at Old Age. Human and Ecological Risk Assessment: Vol. 7, No. 6, pp. 1619-1650. Citation Format: Xiang Gu, LuZhe Sun. Identification of genes with age-dependent expressions in breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1506.