In vivo evidence supporting a metastasis suppressor role for Stard13 (Dlc2) in ErbB2 (Neu) oncogene induced mouse mammary tumors.

Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in the acquisition of the malignant phenotype. The erb-b2 receptor tyrosine kinase 2 (ERBB-2) proto-oncogene is overexpressed in 20-30% of human breast cancers. The StAR related lipid transfer domain containing 13 gene (STARD13), also known as Deleted in Liver Cancer-2 (DLC-2), maps to chromosome band 13q12.3 and is frequently downregulated in human cancers, including 72% of breast cancers. It encodes a RhoGAP protein with sterile α motif (SAM) and StAR-related lipid transfer (START) domains. The objective of this study was to determine if loss of Stard13 plays a role in mammary tumor progression using transgenic mice expressing the activated ErbB-2 (Neu) oncogene and Cre recombinase (NIC) in mammary epithelium under transcriptional control of the murine mammary tumor virus (MMTV) promoter (MMTV-NIC). These mice were crossed with a conditional Stard13 knockout mouse (floxed exon 3), resulting in simultaneous Neu expression and Stard13 deletion, specifically in the mammary epithelium. We found that loss of Stard13 did not alter tumor growth nor significantly modify overall survival and tumor free survival. However, there was an increase in the total number of lung metastases in the Stard13 heterozygous or homozygous mice compared with the parental MMTV-NIC strain. Altogether our results indicate that Stard13 acts as a metastasis suppressor rather than a tumor suppressor gene, in Neu oncogene induced mammary tumorigenesis.

DeBouganin Diabody Fusion Protein Overcomes Drug Resistance to ADCs Comprised of Anti-Microtubule Agents.

Antibody drug conjugates (ADC), comprised of highly potent small molecule payloads chemically conjugated to a full-length antibody, represent a growing class of therapeutic agents. The targeting of cytotoxic payloads via the specificity and selectivity of the antibody has led to substantial clinical benefits. However, ADC potency can be altered by mechanisms of resistance such as overexpression of efflux pumps or anti-apoptotic proteins. DeBouganin is a de-immunized variant of bouganin, a ribosome-inactivating protein (RIP) that blocks protein synthesis, thereby leading to apoptosis. When conjugated to trastuzumab (T-deB), deBouganin was more potent than ado-trastuzumab-emtansine (T-DM1) and unaffected by resistance mechanisms to which DM1 is susceptible. To further highlight the differentiating mechanism of action of deBouganin, HCC1419 and BT-474 tumor cells that survived T-DM1 or trastuzumab-MMAE (T-MMAE) treatment were treated with an anti-HER2 C6.5 diabody-deBouganin fusion protein or T-deB. C6.5 diabody-deBouganin and T-deB were potent against HCC1419 and BT-474 cells that were resistant to T-DM1 or T-MMAE killing. The resistant phenotype involved MDR pumps, Bcl-2 family members, and the presence of additional unknown pathways. Overall, the data suggest that deBouganin is effective against tumor cell resistance mechanisms selected in response to ADCs composed of anti-microtubule payloads.

Trastuzumab-deBouganin Conjugate Overcomes Multiple Mechanisms of T-DM1 Drug Resistance.

The development of antibody drug conjugates has provided enhanced potency to tumor-targeting antibodies by the addition of highly potent payloads. In the case of trastuzumab-DM1 (T-DM1), approved for the treatment of metastatic breast cancer, the addition of mertansine (DM1) to trastuzumab substantially increased progression-free survival. Despite these improvements, most patients eventually relapse due to complex mechanisms of resistance often associated with small molecule chemotherapeutics. Therefore, identifying payloads with different mechanisms of action (MOA) is critical for increasing the efficacy of targeted therapeutics and ultimately improving patient outcomes. To evaluate payloads with different MOA, deBouganin, a deimmunized plant toxin that inhibits protein synthesis, was conjugated to trastuzumab and compared with T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated greater potency in vitro against most cells lines with high levels of Her2 expression. In addition, T-deB, unlike T-DM1, was unaffected by inhibitors of multidrug resistance, Bcl-2-mediated resistance, or Her2-Her3 dimerization. Contrary to T-DM1 that showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell properties. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin could be a highly effective against tumor cell phenotypes not being addressed by current antibody drug conjugate formats and thereby provide prolonged clinical benefit.

Human Leukocyte Antigen-Presented Macrophage Migration Inhibitory Factor Is a Surface Biomarker and Potential Therapeutic Target for Ovarian Cancer.

T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy.

Distinct biological roles for the akt family in mammary tumor progression.

The phosphatidylinositol 3' kinase/Akt pathway is frequently dysregulated in cancer, which can have unfavorable consequences in terms of cell proliferation, survival, metabolism, and migration. Increasing evidence suggests that Akt1, Akt2, and Akt3 play unique roles in breast cancer initiation and progression. We have recently shown that in contrast to Akt1, which accelerates mammary tumor induction in transgenic mice, Akt2 promotes metastasis of tumor cells without affecting the latency of tumor development. Despite the distinct phenotypic outputs resulting from Akt1 or Akt2 activation, very little is known about the mode by which such unique functions originate from these highly related kinases. Here we discuss potential mechanisms contributing to the differing functional specificity of Akt1 and Akt2 with respect to migration, invasion, and metastasis.

Akt1 and akt2 play distinct roles in the initiation and metastatic phases of mammary tumor progression.

The phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway is often dysregulated in cancer. Our previous studies have shown that coexpression of activated Akt1 with activated ErbB2 or polyoma virus middle T antigen uncoupled from the PI3K pathway (PyVmT Y315/322F) accelerates mammary tumor development but cannot rescue the metastatic phenotype associated with these models. Here, we report the generation of transgenic mice expressing activated Akt2 in the mammary epithelium. Like the mouse mammary tumor virus-Akt1 strain, mammary-specific expression of Akt2 delayed mammary gland involution. However, in contrast to Akt1, coexpression of Akt2 with activated ErbB2 or PyVmT Y315/322F in the mammary glands of transgenic mice did not affect the latency of tumor development. Strikingly, Akt2 coexpresssion markedly increased the incidence of pulmonary metastases in both tumor models, demonstrating a unique role in tumor progression. Together, these observations argue that these highly conserved kinases have distinct biological and biochemical outputs that play opposing roles in mammary tumor induction and metastasis.

An EGR2/CITED1 transcription factor complex and the 14-3-3sigma tumor suppressor are involved in regulating ErbB2 expression in a transgenic-mouse model of human breast cancer.

Amplification and elevated expression of the ErbB2 receptor tyrosine kinase occurs in 20% of human breast cancers and is associated with a poor prognosis. We have previously demonstrated that mammary tissue-specific expression of activated ErbB2 under the control of its endogenous promoter results in mammary tumor formation. Tumor development was associated with amplification and overexpression of ErbB2 at both the transcript and protein levels. Here we demonstrate that the EGR2/Krox20 transcription factor and its coactivator CITED1 are coordinately upregulated during ErbB2 tumor induction. We have identified an EGR2 binding site in the erbB2 promoter and demonstrated by chromatin immunoprecipitation assays that EGR2 and CITED1 associate specifically with this region of the promoter. EGR2 and CITED1 were shown to associate, and expression from an erbB2 promoter-reporter construct was stimulated by EGR2 and was further enhanced by CITED1 coexpression. Furthermore, expression of the 14-3-3sigma tumor suppressor led to downregulation of ErbB2 protein levels and relocalization of EGR2 from the nucleus to the cytoplasm. Taken together, these observations suggest that, in addition to an increased gene copy number and upregulation of EGR2 and CITED1, an elevated erbB2 transcript level involves the loss of 14-3-3sigma, which sequesters a key transcriptional regulator of the erbB2 promoter.