The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells.

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.

Monocytes promote liver carcinogenesis in an oncogene-specific manner.

BACKGROUND & AIMS: The leukocyte composition of tumors is heterogeneous, as is the involvement of each leukocyte subset in promoting or restraining tumorigenesis. This heterogeneity reflects the tissue of origin, tumor stage, and the functional state of leukocyte activation, but its biological roots remain poorly understood. Since tumorigenesis is driven by various genetic events, we assessed the role of driver genes in shaping the profiles and the roles of leukocytes in tumorigenesis. METHODS: Mouse liver tumors were induced by hepatic overexpression of either MYC or the combination of myristoylated AKT and NRAS(V12) oncogenes via hydrodynamic transfection. A comparative, flow cytometry- and histology-based immunophenotyping of liver-infiltrating leukocytes was performed at various stages of liver tumorigenesis. The roles of the most abundant leukocyte subsets in tumorigenesis were addressed by immunodepletion. The contribution of liver injury was assessed by comparing the injury-inducing hydrodynamic transfection model to a model in which MYC is an inducible transgene. RESULTS: Myristoylated AKT and NRAS(V12) promoted a marked recruitment of CD11b(+)Ly6G(hi)Ly6C(int) neutrophils and CD11b(+)Ly6G(-)Ly6C(hi) monocytes to the liver, but their immunodepletion did not alter tumorigenesis. In contrast, despite minimal invasion by monocytes/neutrophils during MYC-driven tumorigenesis, immunodepletion of these cells reduced MYC tumor burden and extended survival. MYC-driven tumor initiation was augmented specifically by Ly6C+ monocytes and their ability to promote liver injury. CONCLUSIONS: Our results demonstrate that leukocyte profiles do not necessarily predict their involvement in tumorigenesis, the functional role of leukocytes can be shaped by oncogenes, and that monocyte-dependent tissue injury selectively cooperates with MYC during tumorigenesis.

Reciprocal interaction of Wnt and RXR-α pathways in hepatocyte development and hepatocellular carcinoma.

Genomic analysis of human hepatocellular carcinoma (HCC) is potentially confounded by the differentiation state of the hepatic cell-of-origin. Here we integrated genomic analysis of mouse HCC (with defined cell-of-origin) along with normal development. We found a major shift in expression of Wnt and RXR-α pathway genes (up and down, respectively) coincident with the transition from hepatoblasts to hepatocytes. A combined Wnt and RXR-α gene signature categorized HCCs into two subtypes (high Wnt, low RXR-α and low Wnt, high RXR-α), which matched cell-of-origin in mouse models and the differentiation state of human HCC. Suppression of RXR-α levels in hepatocytes increased Wnt signaling and enhanced tumorigenicity, whereas ligand activation of RXR-α achieved the opposite. These results corroborate that there are two main HCC subtypes that correspond to the degree of hepatocyte differentation and that RXR-α, in part via Wnt signaling, plays a key functional role in the hepatocyte-like subtype and potentially could serve as a selective therapeutic target.

Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma.

Sox9 has gained increasing importance both functionally and as a prognostic factor in cancer. We demonstrate a functional role for Sox9 in inducing a mesenchymal phenotype in lung ADC. We show that Sox9 mRNA and protein are overexpressed in lung ADC, particularly those with KRAS mutations. Sox9 expression correlated with the Notch target gene Hes1, and numerous other Notch pathway components. We observed that Sox9 is a potent inducer of lung cancer cell motility and invasion, and a negative regulator of E-cadherin, a key protein that is lost during epithelial-mesenchymal transition (EMT). Moreover, we show that Notch1 signaling directly regulates Sox9 expression through a SOX9 promoter binding site, independently of the TGF-ß pathway, and that Sox9 participates in Notch-1 induced cell motility, cell invasion, and loss of E-cadherin expression. Together, the results identify a new functional role for a Notch1-Sox9 signaling axis in lung ADC that may explain the correlation of Sox9 with tumor progression, higher tumor grade, and poor lung cancer survival. In addition to Notch and TGF-ß, Sox9 also acts downstream of NF-κB, BMP, EGFR, and Wnt/ß-catenin signaling. Thus, Sox9 could potentially act as a hub to mediate cross-talk among key oncogenic pathways in lung ADC. Targeting Sox9 expression or transcriptional activity could potentially reduce resistance to targeted therapy for lung ADC caused by pathway redundancy.

Diamond-lipid hybrids enhance chemotherapeutic tolerance and mediate tumor regression.

Self-assembled nanodiamond-lipid hybrid particles (NDLPs) harness the potent interaction between the nanodiamond (ND)-surface and small molecules, while providing a mechanism for cell-targeted imaging and therapy of triple negative breast cancers. Epidermal growth factor receptor-targeted NDLPs are highly biocompatible particles that provide cell-specific imaging, promote tumor retention of ND-complexes, prevent epirubicin toxicities and mediate regression of triple negative breast cancers.

Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency.

Nucleostemin (NS) is a nucleolar GTP-binding protein that was first identified in neural stem cells, the functions of which remain poorly understood. Here, we report that NS is required for mouse embryogenesis to reach blastulation, maintenance of embryonic stem cell (ESC) self-renewal, and mammary epithelial cell (MEC) reprogramming to induced pluripotent stem (iPS) cells. Ectopic NS also cooperates with OCT4 and SOX2 to reprogram MECs and mouse embryonic fibroblasts to iPS cells. NS promotes ESC self-renewal by sustaining rapid transit through the G1 phase of the cell cycle. Depletion of NS in ESCs retards transit through G1 and induces gene expression changes and morphological differentiation through a mechanism that involves the MEK/ERK protein kinases and that is active only during a protracted G1. Suppression of cell cycle inhibitors mitigates these effects. Our results implicate NS in the maintenance of ESC self-renewal, demonstrate the importance of rapid transit through G1 for this process, and expand the known classes of reprogramming factors.

MYC suppresses cancer metastasis by direct transcriptional silencing of αv and ß3 integrin subunits.

Overexpression of MYC transforms cells in culture, elicits malignant tumours in experimental animals and is found in many human tumours. We now report the paradoxical finding that this powerful oncogene can also act as a suppressor of cell motility, invasiveness and metastasis. Overexpression of MYC stimulated proliferation of breast cancer cells both in culture and in vivo as expected, but inhibited motility and invasiveness in culture, and lung and liver metastases in xenografted tumours. We show further that MYC represses transcription of both subunits of αvß3 integrin, and that exogenous expression of ß3 integrin in human breast cancer cells that do not express this integrin rescues invasiveness and migration when MYC is downregulated. These data uncover an unexpected function of MYC, provide an explanation for the hitherto puzzling literature on the relationship between MYC and metastasis, and reveal a variable that could influence the development of therapies that target MYC.

Oncogene-specific formation of chemoresistant murine hepatic cancer stem cells.

UNLABELLED: At least some cancer stem cells (CSCs) display intrinsic drug resistance that may thwart eradication of a malignancy by chemotherapy. We explored the genesis of such resistance by studying mouse models of liver cancer driven by either MYC or the combination of oncogenic forms of activation of v-akt murine thymoma viral oncogene homolog (AKT) and NRAS. A common manifestation of chemoresistance in CSCs is efflux of the DNA-binding dye Hoechst 33342. We found that only the MYC-driven tumors contained a subset of cells that efflux Hoechst 33342. This "side population" (SP) was enriched for CSCs when compared to non-SP tumor cells and exhibited markers of hepatic progenitor cells. The SP cells could differentiate into non-SP tumor cells, with coordinate loss of chemoresistance, progenitor markers, and the enrichment for CSCs. In contrast, non-SP cells did not give rise to SP cells. Exclusion of Hoechst 33342 is mediated by ATP binding cassette drug transporter proteins that also contribute to chemoresistance in cancer. We found that the multidrug resistance gene 1 (MDR1) transporter was responsible for the efflux of Hoechst from SP cells in our MYC-driven model. Accordingly, SP cells and their tumor-initiating subset were more resistant than non-SP cells to chemotherapeutics that are effluxed by MDR1. CONCLUSION: The oncogenotype of a tumor can promote a specific mechanism of chemoresistance that can contribute to the survival of hepatic CSCs. Under circumstances that promote differentiation of CSCs into more mature tumor cells, the chemoresistance can be quickly lost. Elucidation of the mechanisms that govern chemoresistance in these mouse models may illuminate the genesis of chemoresistance in human liver cancer.

The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type.

The altered metabolism of tumors has been considered a target for anticancer therapy. However, the relationship between distinct tumor-initiating lesions and anomalies of tumor metabolism in vivo has not been addressed. We report that MYC-induced mouse liver tumors significantly increase both glucose and glutamine catabolism, whereas MET-induced liver tumors use glucose to produce glutamine. Increased glutamine catabolism in MYC-induced liver tumors is associated with decreased levels of glutamine synthetase (Glul) and the switch from Gls2 to Gls1 glutaminase. In contrast to liver tumors, MYC-induced lung tumors display increased expression of both Glul and Gls1 and accumulate glutamine. We also show that inhibition of Gls1 kills cells that overexpress MYC and catabolize glutamine. Our results suggest that the metabolic profiles of tumors are likely to depend on both the genotype and tissue of origin and have implications regarding the design of therapies targeting tumor metabolism.

Activated Notch1 induces lung adenomas in mice and cooperates with Myc in the generation of lung adenocarcinoma.

Notch1 encodes the canonical member of the mammalian Notch receptor family. Activating lesions frequently affect Notch1 in T-cell acute lymphoblastic leukemia (T-ALL) and, recently, have been found in non-small-cell lung cancer (NSCLC) as well. We explored the oncogenic potential of activated Notch1 in the lung by developing a transgenic mouse model in which activated Notch1 was overexpressed in the alveolar epithelium. The initial response to activated Notch1 was proliferation and the accumulation of alveolar hyperplasia, which was then promptly cleared by apoptosis. After an extended latency period, however, pulmonary adenomas appeared in the transgenic mice but failed to progress to become carcinomas. Interestingly, Myc and MycL1 were expressed in the adenomas, suggesting that selection for enhanced Myc activity may facilitate tumorigenesis. Using mice engineered to coexpress activated Notch1 and Myc, we found that supplementing Myc expression resulted in increased frequency of Notch1 intracellular domain (N1ICD)-induced adenoma formation and enabled progression to adenocarcinoma and metastases. Cooperation stemmed from synergistic activation of tumor cell cycling, a process that apparently countered any impedance to tumorigenesis posed by Myc and/or activated Notch1-induced apoptosis. Significantly, cooperation was independent of RAS activation. Taken together, the data suggest that activated Notch1 substitutes for RAS activation synergistically with Myc in the development of NSCLC. These tumor models should be valuable for exploring the role of activated Notch1 in the genesis of NSCLC and for testing therapies targeting either activated Notch1 or its downstream effectors.

Interaction between MYC and MCL1 in the genesis and outcome of non-small-cell lung cancer.

MYC exerts both positive and negative functions in cancer cells, such that its procancerous effects are unmasked only after its anticancer effects are blocked. Here we used multiple mouse models of lung adenocarcinoma to identify genetic events that can cooperate with MYC activation to promote the genesis of non-small-cell lung cancer (NSCLC), the most common form of lung cancer in humans. MYC overexpression targeted to pulmonary alveolar cells was sufficient to induce lung adenomas and carcinomas. Tumorigenesis was assisted by either spontaneous mutations in Kras or experimental introduction of activated RAS, but investigations revealed that additional events were required to circumvent apoptosis, one of the most significant negative functions exerted by MYC. We determined that overexpression of the antiapoptotic protein MCL1 was sufficient to circumvent apoptosis in this setting. Previous clinical studies have indicated that prognosis of human NSCLC is not associated with MCL1, despite its overexpression in many NSCLCs. In reexamining the prognostic value in this setting, we found that MCL1 overexpression does correlate with poor patient survival, but only when accompanied by MYC overexpression. Our findings therefore produce a convergence of mouse and human results that explain how MCL1 can block an important negative consequence of MYC overexpression in both experimental models and clinical cases of NSCLC.

A new transgenic mouse line for tetracycline inducible transgene expression in mature melanocytes and the melanocyte stem cells using the Dopachrome tautomerase promoter.

We have generated a novel transgenic mouse to direct inducible and reversible transgene expression in the melanocytic compartment. The Dopachrome tautomerase (Dct) control sequences we used are active early in the development of melanocytes and so this system was designed to enable the manipulation of transgene expression during development in utero and in the melanocyte stem cells as well as mature melanocytes. We observed inducible lacZ and GFP reporter transgene activity specifically in melanocytes and melanocyte stem cells in mouse skin. This mouse model will be a useful tool for the pigment cell community to investigate the contribution of candidate genes to normal melanocyte and/or melanoma development in vivo. Deregulated expression of the proto-oncogene MYC has been observed in melanoma, however whether MYC is involved in tumorigenesis in pigment cells has yet to be directly investigated in vivo. We have used our system to over-express MYC in the melanocytic compartment and show for the first time that increased MYC expression can indeed promote melanocytic tumor formation.

Redefining the expression and function of the inhibitor of differentiation 1 in mammary gland development.

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation.

Therapeutic potential of a synthetic lethal interaction between the MYC proto-oncogene and inhibition of aurora-B kinase.

The Myc protein and proteins that participate in mitosis represent attractive targets for cancer therapy. However, their potential is presently compromised by the threat of side effects and by a lack of pharmacological inhibitors of Myc. Here we report that a circumscribed exposure to the aurora kinase inhibitor, VX-680, selectively kills cells that overexpress Myc. This synthetic lethal interaction is attributable to inhibition of aurora-B kinase, with consequent disabling of the chromosomal passenger protein complex (CPPC) and ensuing DNA replication in the absence of cell division; executed by sequential apoptosis and autophagy; not reliant on the tumor suppressor protein p53; and effective against mouse models for B-cell and T-cell lymphomas initiated by transgenes of MYC. Our findings cast light on how inhibitors of aurora-B kinase may kill tumor cells, implicate Myc in the induction of a lethal form of autophagy, indicate that expression of Myc be a useful biomarker for sensitivity of tumor cells to inhibition of the CPPC, dramatize the virtue of bimodal killing by a single therapeutic agent, and suggest a therapeutic strategy for killing tumor cells that overexpress Myc while sparing normal cells.

A dominant mutant allele of the ING4 tumor suppressor found in human cancer cells exacerbates MYC-initiated mouse mammary tumorigenesis.

ING4 is a candidate tumor suppressor gene that is deleted in 10% to 20% of human breast cancers and is mutated in various human cancer cell lines. To evaluate whether ING4 has a tumor-suppressive role in breast tissue, we overexpressed it in mouse mammary glands using a transplant system. Ectopic expression of ING4 suppressed MYC-induced mammary hyperplasia, but not tumorigenesis. In the same model system, we show that a COOH-terminal truncation mutant of ING4 found in human cancer cells could act alone to induce abnormal gland structures resembling mammary hyperplasia, which did not progress to tumors. However, coexpression of the ING4 mutant with MYC increased the penetrance and metastasis of MYC-initiated mammary tumors, giving rise to tumors with more organized acinar structures. Similarly, in vitro expression of the ING4 mutant in MCF10A mammary epithelial cells reinforced tight junctional structures. Our results provide direct functional evidence that ING4 could suppress the early stages of breast cancer and that dominant mutant alleles of ING4 might contribute to malignant development.

The B cell antigen receptor and overexpression of MYC can cooperate in the genesis of B cell lymphomas.

A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR) in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL), whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL). We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.

Farnesyl transferase inhibitors induce extended remissions in transgenic mice with mature B cell lymphomas.

BACKGROUND: We have used a mouse model based on overexpression of c-Myc in B cells genetically engineered to be self-reactive to test the hypothesis that farnesyl transferase inhibitors (FTIs) can effectively treat mature B cell lymphomas. FTIs are undergoing clinical trials to treat both lymphoid and non-lymphoid malignancies and we wished to obtain evidence to support the inclusion of B cell lymphomas in future trials. RESULTS: We report that two FTIs, L-744,832 and SCH66336, blocked the growth of mature B cell lymphoma cells in vitro and in vivo. The FTI treatment affected the proliferation and survival of the transformed B cells to a greater extent than naïve B cells stimulated with antigen. In syngeneic mice transplanted with the transgenic lymphoma cells, L-744,832 treatment prevented the growth of the tumor cells and the morbidity associated with the resulting lymphoma progression. Tumors that arose from transplantation of the lymphoma cells regressed with as little as three days of treatment with L-744,832 or SCH66336. Treatment of these established lymphomas with L-744,832 for seven days led to long-term remission of the disease in approximately 25% of animals. CONCLUSION: FTI treatment can block the proliferation and survival of self-reactive transformed B cells that overexpress Myc. In mice transplanted with mature B cell lymphomas, we found that FTI treatment led to regression of disease. FTIs warrant further consideration as therapeutic agents for mature B cell lymphomas and other lymphoid tumors.

Id1 cooperates with oncogenic Ras to induce metastatic mammary carcinoma by subversion of the cellular senescence response.

Recent evidence demonstrates that senescence acts as a barrier to tumorigenesis in response to oncogene activation. Using a mouse model of breast cancer, we tested the importance of the senescence response in solid cancer and identified genetic pathways regulating this response. Mammary expression of activated Ras led to the formation of senescent cellular foci in a majority of mice. Deletion of the p19(ARF), p53, or p21(WAF1) tumor suppressors but not p16(INK4a) prevented senescence and permitted tumorigenesis. Id1 has been implicated in the control of senescence in vitro, and elevated expression of Id1 is found in a number of solid cancers, so we tested whether overexpression of Id1 regulates senescence in vivo. Although overexpression of Id1 in the mammary epithelium was not sufficient for tumorigenesis, mice with expression of both Id1 and activated Ras developed metastatic cancer. These tumors expressed high levels of p19(Arf), p53, and p21(Waf1), demonstrating that Id1 acts to make cells refractory to p21(Waf1)-dependent cell cycle arrest. Inactivation of the conditional Id1 allele in established tumors led to widespread senescence within 10 days, tumor growth arrest, and tumor regression in 40% of mice. Mice in which Id1 expression was inactivated also exhibited greatly reduced pulmonary metastatic load. These data demonstrate that established tumors remain sensitive to senescence and that Id1 may be a valuable target for therapy.

Distinct pathways of genomic progression to benign and malignant tumors of the liver.

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.