Gene therapy approach prevents BCIC from binding to antiapoptotic proteins.

Blocking a cancer cell protein from binding to three other proteins may provide a new approach to cancer therapy that both reduces populations of breast cancer initiating cells (BCICs) in breast tumors and sensitizes the tumors to existing treatments such as lapatinib or paclitaxel, scientists claim. The technique uses a specially designed lipid-based vector to make cancer cells, including BCICs, express a mutant form of the BH3-only proapoptotic protein (Bik).

The mutant protein, called BikDD, essentially competes with Bik for binding to the three antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. This results in significant antitumor and apoptotic effects and, importantly, improves the anticancer effects of lapatinib or paclitaxel in relevant tumor types, claim the University of Texas M.D. Anderson Cancer Center researchers.

Reporting on their in vitro and in vivo studies in Cancer Cell, Mien-Chie Hung, Ph.D., and colleagues, claim that their results in addition highlight an important role for the antiapoptotic Bcl-2 proteins in the survival of BCICs. Their paper is titled “BikDD Eliminates Breast Cancer Initiating Cells and Synergizes with Lapatinib for Breast Cancer Treatment.”

There are currently no drugs that can effectively reduce BCICs in patients, and resistance of these cells to chemo- and radiotherapies means that following therapy, the relative proportions of these cells in the tumors increase, and eventually lead to relapse, the researchers report.

One of the key mechanisms accounting for chemoresistance in cancer-initiating cells is their low susceptibility to apoptosis, and previous lines of research have implicated the Bcl-2 family of proteins in the ability of cancer cells to escape apoptosis in response to cancer therapy. For example, studies have shown that overexpression of the antiapoptosis proteins Bcl-2, Bcl-xL, and Mcl-1 correlates with high tumor grade, poor patient prognosis, and the development of resistance to chemotherapy.

More specifically, the acquired resistance of breast cancer cells to lapatinib has been linked with overexpression of Bcl-2 and Mcl-1, suggesting that lapatinib-induced apoptosis requires inactivation of antiapoptotic Bcl-2 family proteins.

The Anderson team hypothesized that because the overall expression pattern of Bcl-2, Bcl-xL, and Mcl-1 appears to correlate inversely with apoptotic response following drug treatment, an antagonist that targets all of these antiapoptotic proteins might stand a good chance of acting to reinstate apoptotic pathways in breast cancer cells.

The researchers’ approach to achieving this involved introducing into cancer cells a competitive inhibitor, a mutant form of the Bik protein that normally binds to  to Bcl-2, Bcl-xL, and Mcl-1. To test whether this approach might work, they delivered a lentivirus carrying the BIKDD gene into cells from the human breast cancer line MDA-MB-468. These tests provided confirmation that expression of BikDD significantly inhibited cell growth and resulted in large numbers of apoptotic bodies.

Interestingly, expression of BikDD also reduced the population of CD44+/CD24cells (which have previously been identified as breast cancer stem-type cells) and reduced mamosphere formation in vitro. These results were recapitulated in a different cell line: Infecting BT474 human breast cancer cells with the BikDD vector also led to a reduction in the CD44+/CD24population and of mammosphere formation. Importantly, introducing BikDD into human primary breast tumor samples that had undergone radiation therapy similarly led to significant reductions in the CD44+/CD24cell population, and mammosphere formation. Equivalent results were obtained using primary mouse tumor cells: administration of BikDD led to marked reductions in populations of mouse breast stem cells, and again blocked mammosphere formation.

The team went on to investigate whether BikDD could also inhibit cancer initiation. They infected mamospheres from MDA-MB-468 parental cells using the BikDD vector, and then injected surviving cells into NOD/SCID mice. Compared with untreated MDA-MB-468 cells, which readily formed tumors, the BikDD-infected cells demonstrated much lower cancer-forming capacity in vivo, and virtually no tumors developed in the recipient animals, suggesting that BikDD treatment reduced the BCIC population, the researchers remark.

They then adopted a gene therapy protocol that allows for the assay of cancer initiation activity in tumor xenografts growing in mice after BikDD treatment. This approach exploits a cancer cell-targeting platform developed at the MD Anderson Center, called VISA, VISA’ (VP16-GAL4-WPRE integrated systemic amplifier), which is based on an engineered, promotor-driven expression vector designed to enhance cancer-specific promoter activity by several hundred-fold, and prolong duration of gene expression without loss of cancer specificity.

Mice bearing MDA-MB-468 tumor xenografts were treated using either a control vector-liposome or with VISA-claudin4-BikDD-liposome complexes, and resulting tumor tissues removed and subsequently passaged into new animals. The results showed that transplanted cells taken from mice that had been treated with VISAclaudin4-BikDD-liposome complexes were far less tumorigenic in new animals than those from mice treated with vector-control-liposome complexes. In fact, none of the animals given tumor cells from the VISA-claudin4-BikDD-treated mice developed cancers. These animals also demonstrated lower numbers of CD44+/CD24 cells, and fewer mammospheres formed after VISAclaudin4-BikDD treatment.

Because the team’s previous work had suggested that in comparison with wild-type Bik, BikDD demonstrates enhanced binding affinity to Bcl-2 antiapoptotic proteins, they looked more specifically at the effect of its major binding partners Bcl-2, Bcl-xL, and Mcl-1, in BCICs. Using combinations of shRNAs to silence the three Bcl-2, Bcl-xL, and Mcl-1 either individually or in combinations in cultured cells, the researchers found that while knocking down any of the proteins individually had no effect on the numbers of BCIC cells, silencing all three simultaneously reduced the CD44+/CD24population to 25% of that in control MDA-MB-468 cells, and consequently decreased mammosphere formation. Similar results were obtained using different shRNAs (to verify that the effects weren’t due to off-target activity), and in a different cell line.

“Taken together, we determined that efficient induction of apoptosis in BCICs requires silencing of all three antiapoptotic Bcl-2 proteins, which suggests that co-antagonism of multiple Bcl-2 antiapoptotic proteins by BikDD may have a better killing effect against BCICs than targeting individual antiapoptotic proteins, which is likely due to their functional redundancy in the survival of BCICs,” the authors state.

They then exploited the cancer cell-targeting VISA technology to test the therapeutic effects of BikDD gene therapy both in vitro and in vivo. To this end, they engineered a VISA vector that would express BikDD under the claudin-4 promoter that is selectively expressed in breast cancer cells. Testing the resulting VISA-claudin4–BikDD vector in a panel of breast cancer and normal cell lines confirmed that it strongly inhibited the growth of different breast cancer cell lines, but had little or no effect on the growth of normal human cells. The tumor inhibitory effects of the vector were subsequently confirmed in vivo, in one syngeneic mouse breast tumor and multiple human breast tumor orthotopic xenograft models.

Prior studies had demonstrated that the clinical efficacy of anti-Her2 drugs such as lapatinib and trastuzumab are greatly limited by either inoperative apoptosis machinery or overexpression of Bcl-2 antiapoptotic proteins, the researchers add. With this in mind they moved on to examine whether either the administration of BikDD, or the inhibition of antiapoptotic Bcl-2 proteins could enhance the therapeutic effect of lapatinib in breast cancer cells. They found that VISA-claudin4-BikDD effectively sensitized BT474 and MDA-MB-453 (Her2+), and MDA-MB-468 and BT20 (EGFR+) cells to lapatinib. Similarly, inhibiting Bcl-2, Bcl-xL, and Mcl-1 using shRNAs also sensitized EGFR+/Her2+ breast cancer cells to lapatinib, to about the same degree as BikDD vector therapy. Significantly, VISA-claudin4-BikDD therapy in addition sensitized multiple breast cancer cell lines to paclitaxel in vitro.

To further examine the therapeutic efficacy of VISA-claudin4-BikDD plus lapatinib combination in vivo, the researchers then treated mice bearing Her2+ BT474 human breast cancer xenografts, with VISA-claudin4-BikDD and/or lapatinib. While VISA-claudin4-BikDD or lapatinib alone had significant tumor inhibitory effects, combining the two treatments demonstrated even better therapeutic efficacy. These results were confirmed in mice carrying tumors derived from different breast cancer cell lines.

To evaluate therapy on BCIC cells in vivo, VISA-claudin4-BikDD, lapatinib, or paclitaxel were either alone or in combination, to treat a MDA-MB-468 tumor orthotopic xenograft mouse model. Consistent with the in vitro data, BikDD treatment significantly reduced the percentage of CD44+/CD24 cells, whereas, as expected, paclitaxel therapy on its own increased this population by about threefold. In fact, combining the two treatments was better at suppressing tumor growth than VISA-claudin3-BikDD therapy alone, even after therapy was withdrawn, the authors note. Similar results were observed as a result of combination therapy with VISAclaudin4-BikDD and lapatinib.

Collectively, these results indicate that BikDD driven by VISA-claudin4 vector potently reduced the CD44+/CD24 population in vivo even after chemotherapy, and efficiently attenuated tumor growth after cessation of drug treatment, suggesting that VISA-claudin4-BikDD treatment may serve as a potential therapeutic approach to kill BCICs, which is considered as a major barrier for breast cancer treatment,” the authors write. “By using our newly developed VISA-claudin4-BikDD for treating breast cancer, it is likely that therapeutic efficacy will be enhanced and potential side effects prevented as we have shown that BikDD targets both non-BCICs and BCICs and demonstrates virtually no toxicity in normal cells…Therefore, it is worthy of moving VISA-claudin4-BikDD into a clinical trial.”

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