Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells

Abstract

BackgroundIn normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery.ResultsAAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis.ConclusionAAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.

Adeno-associated virus type 2 infection of nude mouse human breast cancer xenograft induces necrotic death and inhibits tumor growth

“…16 We showed that AAV2 induced caspase-independent apoptosis in MCF-7 (ER + ) cells, caspase-dependent apoptosis in MDA-MB-468 (ER -) cells and caspase-dependent apoptosis, mixed with necrosis, in MDA-MB-231 (ER -) cells. 16 A 100% decrease in viability of the AAV2-infected breast cancer cell lines was correlated with the ability of the virus to undergo active genome replication and differentially express its non-structural proteins: Rep78, Rep68, and Rep40, but not Rep52.…”

“…16 We showed that AAV2 induced caspase-independent apoptosis in MCF-7 (ER + ) cells, caspase-dependent apoptosis in MDA-MB-468 (ER -) cells and caspase-dependent apoptosis, mixed with necrosis, in MDA-MB-231 (ER -) cells. 16 A 100% decrease in viability of the AAV2-infected breast cancer cell lines was correlated with the ability of the virus to undergo active genome replication and differentially express its non-structural proteins: Rep78, Rep68, and Rep40, but not Rep52. Although our studies failed to identify common AAV2-targeted cell death pathways among the different breast cancer cell lines, our studies did show that cellular demise coincided with increased expression of c-Myc and Ki-67 protein levels, irrespective of the breast cancer cell line studied.…”

“…19,20 We hypothesized that the inherent endonuclease activity of AAV2 Rep78 and Rep68 proteins induces a DNA damage response and activates death signaling pathways, as well as simultaneously mediate conflicting activation of cell proliferation pathways, which culminates in cancer cell death. 16 In contrast, normal human mammary epithelial cells infected with AAV2 failed to express Rep proteins or undergo apoptosis. 16 Our studies suggest that the mechanistic ability of AAV2 to distinguish between normal and cancer cells to mediate the cell killing could be clinically advantageous.…”

“…16 In contrast, normal human mammary epithelial cells infected with AAV2 failed to express Rep proteins or undergo apoptosis. 16 Our studies suggest that the mechanistic ability of AAV2 to distinguish between normal and cancer cells to mediate the cell killing could be clinically advantageous.…”

“…Although our studies failed to identify common AAV2-targeted cell death pathways among the different breast cancer cell lines, our studies did show that cellular demise coincided with increased expression of c-Myc and Ki-67 protein levels, irrespective of the breast cancer cell line studied. 16 The upregulated expression of c-Myc was a significant observation since this protein has been shown to have the dual ability to induce cell cycle progression/proliferation as well as apoptosis, in the context of other cellular signals. 17,18 In contrast, expression of Ki-67 is a typical marker of proliferation indicative of active S phase cell cycle progression.…”