Xueli Fan

Tumor microenvironment: The role of the tumor stroma in cancer

The tumor microenvironment, composed of non‐cancer cells and their stroma, has become recognized as a major factor influencing the growth of cancer. The microenvironment has been implicated in the regulation of cell growth, determining metastatic potential and possibly determining location of metastatic disease, and impacting the outcome of therapy. While the stromal cells are not malignant per se, their role in supporting cancer growth is so vital to the survival of the tumor that they have become an attractive target for chemotherapeutic agents. In this review, we will discuss the various cellular and molecular components of the stromal environment, their effects on cancer cell dynamics, and the rationale and implications of targeting this environment for control of cancer. Additionally, we will emphasize the role of the bone marrow‐derived cell in providing cells for the stroma. J. Cell. Biochem. 101: 805–815, 2007.

Spontaneous Expression of Embryonic Factors and p53 Point Mutations in Aged Mesenchymal Stem Cells: A Model of Age-Related Tumorigenesis In Mice

Aging is the single most common risk factor for cancer. Peripheral and marrow-derived stem cells are long lived and are candidate cells for the cancer-initiating cell. Repeated rounds of replication are likely required for accumulation of the necessary genetic mutations. Based on the facts that mesenchymal stem cells (MSC) transform with higher frequency than other cell types, and tumors in aged C57BL/6 mice are frequently fibrosarcomas, we used a genetically tagged bone marrow (BM) transplant model to show that aged mice develop MSC-derived fibrosarcomas. We further show that, with aging, MSCs spontaneously transform in culture and, when placed into our mouse model, recapitulated the naturally occurring fibrosarcomas of the aged mice with gene expression changes and p53 mutation similar to the in vivo model. Spontaneously transformed MSCs contribute directly to the tumor, tumor vasculature, and tumor adipose tissue, recruit additional host BM-derived cells (BMDC) to the area, and fuse with the host BMDC. Unfused transformed MSCs act as the cancer stem cell and are able to form tumors in successive mice, whereas fusion restores a nonmalignant phenotype. These data suggest that MSCs may play a key role in age-related tumors, and fusion with host cells restores a nonmalignant phenotype, thereby providing a mechanism for regulating tumor cell activity.

Interaction of oncogenic papillomavirus E6 proteins with fibulin-1.

Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calcium-binding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6.

Human and Mouse Colon Cancer Utilizes CD95 Signaling for Local Growth and Metastatic Spread to Liver

BACKGROUND & AIMS Analysis of clinical colon cancer specimens show alterations in the CD95 (Fas Ag/Fas L) pathway as tumors progress from local to metastatic disease, suggesting that this pathway may play a role in invasive behavior of colon cancer. However, direct causality between these alterations and clinical disease progression has not been shown. METHODS Surgically resected metastatic colon cancer samples were evaluated for Fas Ag/L and apoptosis. Alterations in the Fas-signaling pathway found in human samples were recreated through a series of staged transfection experiments in the MC38 mouse colon cancer cell line and the effects on growth tested in vitro and in vivo. RESULTS Expression of FLICE-like inhibitory protein confers apoptosis resistance, increasing the incidence of primary tumors through a survival advantage by avoiding apoptosis and inducing Fas-mediated proliferation. Coexpression of Fas L enables colon cancer cells to metastasize to the liver from local tumors as well as from intravenous injection of cells. MC38-FasL/FLICE-like inhibitory protein colon cancer cells induce apoptosis in hepatocytes via activation of type II Fas Ag signaling, thus creating a niche conducive to tumor growth and fueling their own growth via Fas proliferative signaling. CONCLUSIONS Alterations in the Fas Ag pathway which inhibit apoptosis and increase Fas-mediated proliferation directly increase local colon cancer growth, and enhance metastasis to the liver. Delineating points in the pathway responsible for growth and metastasis will offer targets that may be exploited for therapy.

Mutations in bone marrow-derived stromal stem cells unmask latent malignancy

Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to malignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk tissue. To address this question, Apc(Min/+) and Apc(Min/+) Rag2(-/-) mice, which have a predilection to mammary carcinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC) transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the marrow cavity where they could be recovered up to a year later without apparent effect on the health of the mouse. No mammary tumors were found. However, in mice carrying the Apc(Min/+) mutation, p53MSC homed to mammary tissue and significantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-alpha-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent disease. The increased cancer phenotype was completely preventable with neutralization of TNF-alpha or by transfer of CD4(+) regulatory T cells from immune competent donors, demonstrating that immune competency to regulate inflammation was sufficient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and stromal mutations. The significant synergy between host immunity and mesenchymal cells identified here may restructure treatments to restore an anticancer microenvironment.

T-bet Knockout Prevents Helicobacter felis-Induced Gastric Cancer

Helicobacter infection is the primary risk factor for gastric cancer, with the cytokine environment within the gastric mucosa the strongest predictor of disease risk. Elevated TNF-α, IL-1β, and low IL-10 are associated with the highest risk. In this study, we used C57BL/6 mice to identify T-bet as a central regulator of the cytokine environment during Helicobacter felis infection. We infected male and female C57BL/6 and C57BL/6-T-bet knockout (KO) liter mates with H. felis and examined the bacterial colonization, immune response, and mucosal damage at varying time points. T-bet KO mice maintained infection for 15 mo at similar levels to wild-type mice. Infection and immune response did not differ between male and female mice. Despite sustained infection, T-bet KO mice respond with a blunted Th1 response associated with preservation of parietal and chief cells and protection from the development of gastric cancer. Unexpectedly, T-bet KO mice develop a gastric environment that would not be expected based on the phenotype of T-bet KO CD4 cells alone. T-bet KO mice respond to H. felis infection with a markedly blunted IL-1β and TNF-α and elevated IL-10 levels. Activity of this one master regulator modulates the expression of the key gastric mucosal cytokines associated with gastric cancer and may be a target for therapy to restore immune balance clinically in patients at risk for gastric cancer.

Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage

ABSTRACT Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G2 checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G2 checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.

Down-regulation of p21 contributes to apoptosis induced by HPV E6 in human mammary epithelial cells

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 gene is essential for the oncogenic potential of HPV. E6 induces cell proliferation and apoptosis in cervical cancer precursor lesions and in cultured cells. Although induction of telomerase and inactivation of the tumor suppressor p53 play important roles for E6 to promote cell growth, the molecular basis of E6-induced apoptosis is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies demonstrated that E6 could in fact sensitize cells to apoptosis. Understanding the mechanism of p53-independent apoptosis is of clinical significance. In the present study, we investigated the mechanism of apoptosis during E6-mediated immortalization of primary human mammary epithelial cell (HMEC). E6 by itself is sufficient to immortalize HMECs and is believed to do so at least in part by activation of telomerase. During the process of E6-mediated HMEC immortalization, an increased apoptosis was observed. Mutational analysis demonstrated that E6-induced apoptosis was distinct from its ability to promote cell proliferation, activate telomerase, or degrade p53. While the known pro-apoptotic E6 target proteins such as Bak or c-Myc did not appear to play an important role, down-regulation of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) by E6 correlated with its ability to induce apoptosis. Ectopic expression of p21 inhibited E6-induced apoptosis. Moreover, a p53 degradation defective E6 mutant was competent for p21 down-regulation and apoptosis induction. The anti-apoptotic function of p21 may not simply be the result of p21-induced growth arrest. These studies demonstrate an E6 activity to down-regulate p21 that is important for induction of apoptosis.

Role of Cdk1 in DNA damage-induced G1 checkpoint abrogation by the human papillomavirus E7 oncogene

The high-risk human papillomavirus (HPV) E7 oncogene abrogates DNA damage-induced G1 checkpoint but the mechanism is not fully understood. The G1 kinase Cdk2 is activated in E7-expressing cells. However, whether Cdk2 is required for E7 to abrogate the G1 checkpoint is not known. Accumulating evidence implicates a role for the mitotic Cdk1 in G1/S phase transition in the absence of Cdk2. We therefore examined the expression and requirement of Cdk1 and Cdk2 in the G1 checkpoint abrogation in E7-expressing cells. Although both Cdk1 and Cdk2 were up-regulated in E7-expressing cells upon DNA damage, down-regulation of Cdk1 but not Cdk2 impairs the ability of E7 to abrogate the G1 checkpoint. Our study thus demonstrated an important role for Cdk1 in bypassing the G1 checkpoint in E7-expressing cells. To understand the mechanism by which E7 activates Cdk1, we examined the transcription factor B-Myb. Our studies demonstrated that downregulation of B-Myb reduced the steady-state level of Cdk1 and induced G1 arrest in E7-expressing cells upon DNA damage. In addition, it remains a mystery how E7 promotes cell cycle progression in the presence of Cdk inhibitor p21. As p21 binds Cdk1 with lower affinity than Cdk2, our results suggest a mechanism by which E7 bypasses the inhibitory effect of p21. Nonetheless, our studies demonstrated that p21 still possessed partial ability to arrest cells at G1 phase in E7-expressing cells. These studies shed light on mechanisms by which HPV E7 modulates cell cycle checkpoint.

Association of cottontail rabbit papillomavirus E6 oncoproteins with the hDlg/SAP97 tumor suppressor.

Papillomaviruses are small DNA viruses that infect epithelial tissues and cause warts. Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The E6 and E7 oncogenes are the only genes consistently expressed in HPV‐positive cervical cancer cells. Cottontail rabbit papillomavirus (CRPV) induces papillomas and carcinomas on cottontail and domestic rabbits and provides an excellent animal model of HPV infection and vaccine development. CRPV encodes three transforming proteins; LE6, SE6, and E7. Each of these proteins is required for papilloma formation. Like HPV E7, the CRPV E7 protein binds to the tumor suppressor pRB. In contrast, unlike HPV E6, the CRPV E6 proteins do not bind the tumor suppressor p53. Although more than a dozen cellular proteins have been identified as HPV E6 interacting proteins, nothing is known about the cellular interacting proteins of CRPV E6s. Here we describe the association of CRPV E6s with hDlg/SAP97, the mammalian homolog of the Drosophila discs large tumor suppressor protein. HPV E6 has previously shown to bind and target hDlg/SAP97 for degradation. Our results demonstrate that both LE6 and SE6 interact with hDlg/SAP97, although their association does not lead to the degradation of hDlg/SAP97. The PDZ domains of hDlg were shown to be sufficient for interaction with CRPV E6 proteins while the C‐terminus of CRPV E6 is essential for the interaction with hDlg. The association of hDlg with SE6 may be important but not sufficient for the transformation of NIH 3T3 cells by SE6. Importantly, a CRPV SE6 mutant defective for papilloma formation did not interact with hDlg. These results suggest that interaction with hDlg/SAP97 plays a role in the biological function of CRPV E6s. J. Cell. Biochem. 94: 1038–1045, 2005.