Purpose: Autophagy is a critical survival pathway for cancer cells under conditions of nutrient or oxygen limitation, or cell stress. As a consequence of antiangiogenic therapy, solid tumors encounter hypoxia induction and imbalances in nutrient supply. We wished to determine the role of autophagy in protection of tumor cells from the effects of antiangiogenic therapy and chemotherapy. We examined the effect of inhibiting autophagy on hypoxic colon cancer cells in vitro and on bevacizumab-and oxaliplatin-treated mouse xenografts in vivo.Experimental Design: The autophagic response to hypoxia and DNA-damaging agents was assessed by fluorescent microscopic imaging, autophagy-related gene expression, and by electron microscopic ultrastructural analysis. Pharmacologic and molecular approaches to autophagy inhibition were taken in a panel of colon cancer cell lines. Mouse xenograft models were treated with combinations of oxaliplatin, bevacizumab, and chloroquine to assess effects on tumor growth reduction and on pharmacodynamic markers of autophagy inhibition.Results: Autophagy was induced in colon cancer models by exposure to both hypoxia and oxaliplatin. Inhibition of autophagy, either with chloroquine or by downregulation of beclin1 or of ATG5, enhanced sensitivity to oxaliplatin under normal and hypoxic conditions in a synergistic manner. Both bevacizumab and oxaliplatin treatments activate autophagy in HT29 murine xenografts. The addition of chloroquine to bevacizumab-based treatment provided greater tumor control in concert with evidence of autophagy inhibition.Conclusions: These findings implicate autophagy as a mechanism of resistance to antiangiogenic therapies and support investigation of inhibitory approaches in the management of this disease.

Cell numbers are regulated by a balance among proliferation, growth arrest, and programmed cell death. A profound example of cell homeostasis, controlled throughout life, is the complex process of blood cell development, yet little is understood about the intracellular mechanisms that regulate blood cell growth arrest and programmed cell death. In this work, using transforming growth factor beta 1 (TGF beta 1)-treated M1 myeloid leukemia cells and genetically engineered M1 cell variants, the regulation of growth arrest and apoptosis was dissected. Blocking of early expression of MyD118, a novel differentiation primary response gene also shown to be a primary response gene induced by TGF beta 1, delayed TGF beta 1-induced apoptosis, demonstrating that MyD118 is a positive modulator of TGF beta 1-mediated cell death. Elevated expression of bcl-2 blocked the TGF beta 1-induced apoptotic pathway but not growth arrest induced by TGF beta 1. Deregulated expression of either c-myc or c-myb inhibited growth arrest and accelerated apoptosis, demonstrating for the first time that c-myb plays a role in regulating apoptosis. In all cases, the apoptotic response was correlated with the level of MyD118 expression. Taken together, these findings demonstrate that the primary response gene MyD118 and the c-myc, c-myb, and bcl-2 proto-oncogenes interact to modulate growth arrest and apoptosis of myeloid cells.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.

Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxiainduced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia (4 h) three times a week. The resulting two hypoxiaconditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxiaconditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient (SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs.

Angiogenesis inhibition has been shown to enhance the therapeutic efficacy of cytotoxic chemotherapy in colorectal cancer. The basis of the contribution of this modality has not been defined fully. To determine the potential role of hypoxia-induced apoptosis, we studied a series of colon cancer cell lines with varying susceptibility to hypoxia. We exposed HT29 and HCT116 colon adenocarcinoma cell lines to sublethal periods of hypoxia three times weekly for 40 exposures, and derived cell lines both more resistant (from HT29) and more sensitive (from HCT116) to hypoxia-induced apoptosis. Both hypoxia-derived cell lines demonstrated more rapid growth than the parental lines when implanted subcutaneously in immunodeficient mice. Treatment of tumor-bearing mice with bevacizumab resulted in depletion of tumor microvasculature, upregulation of Hypoxia-inducible factor-1 alpha (HIF-1alpha), and increased pimonidazole staining, consistent with an anti-angiogenic effect and induction of hypoxia in tumors derived from all cell lines. The proportion of apoptotic cells was increased in all the treated tumors, and was most pronounced in the bevacizumab-treated HCT116-derived cells. The bevacizumab-treated tumors showed growth delay in HT29 and its derivative, and the parental HCT116. In the hypoxia-sensitive HCT116-derived tumors, marked tumor shrinkage and prolonged growth control occurred. Therefore, bevacizumab treatment is an effective inducer of a hypoxic environment, but the resulting cell kill and tumor shrinkage is determined by the susceptibility of the tumor to apoptosis. The induction of apoptosis by hypoxia may contribute to the benefits of such treatment in the clinical setting.