Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics.

Resistance to anti-tumor therapeutics is an important clinical problem. Tumor-targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a "Trojan-Horse" approach to combating these tumors by using a receptor-targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non-covalent self-assembly with therapeutic cargo, forming HER3-homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re-introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here.

Evaluation of venous thrombosis and tissue factor in epithelial ovarian cancer.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population. METHODS: We performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF. RESULTS: 35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75-6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001. CONCLUSIONS: TFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.

Sphingosine kinase 1 is required for TGF-ß mediated fibroblastto- myofibroblast differentiation in ovarian cancer.

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-ß-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-ß signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

Suboptimal cytoreduction in ovarian carcinoma is associated with molecular pathways characteristic of increased stromal activation.

OBJECTIVE: Suboptimal cytoreductive surgery in advanced epithelial ovarian cancer (EOC) is associated with poor survival but it is unknown if poor outcome is due to the intrinsic biology of unresectable tumors or insufficient surgical effort resulting in residual tumor-sustaining clones. Our objective was to identify the potential molecular pathway(s) and cell type(s) that may be responsible for suboptimal surgical resection. METHODS: By comparing gene expression in optimally and suboptimally cytoreduced patients, we identified a gene network associated with suboptimal cytoreduction and explored the biological processes and cell types associated with this gene network. RESULTS: We show that primary tumors from suboptimally cytoreduced patients express molecular signatures that are typically present in a distinct molecular subtype of EOC characterized by increased stromal activation and lymphovascular invasion. Similar molecular pathways are present in EOC metastases, suggesting that primary tumors in suboptimally cytoreduced patients are biologically similar to metastatic tumors. We demonstrate that the suboptimal cytoreduction network genes are enriched in reactive tumor stroma cells rather than malignant tumor cells. CONCLUSION: Our data suggest that the success of cytoreductive surgery is dictated by tumor biology, such as extensive stromal reaction and increased invasiveness, which may hinder surgical resection and ultimately lead to poor survival.

Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC.

Chemotherapy targeting by DNA capture in viral protein particles.

AIM: This study tests the hypothesis that DNA intercalation and electrophilic interactions can be exploited to noncovalently assemble doxorubicin in a viral protein nanoparticle designed to target and penetrate tumor cells through ligand-directed delivery. We further test whether this new paradigm of doxorubicin targeting shows therapeutic efficacy and safety in vitro and in vivo. MATERIALS & METHODS: We tested serum stability, tumor targeting and therapeutic efficacy in vitro and in vivo using biochemical, microscopy and cytotoxicity assays. RESULTS: Self-assembly formed approximately 10-nm diameter serum-stable nanoparticles that can target and ablate HER2+ tumors at >10× lower dose compared with untargeted doxorubicin, while sparing the heart after intravenous delivery. The targeted nanoparticle tested here allows doxorubicin potency to remain unaltered during assembly, transport and release into target cells,while avoiding peripheral tissue damage and enabling lower, and thus safer, drug dose for tumor killing. CONCLUSION: This nanoparticle may be an improved alternative to chemical conjugates and signal-blocking antibodies for tumor-targeted treatment.

Xeroderma pigmentosum complementation group C single-nucleotide polymorphisms in the nucleotide excision repair pathway correlate with prolonged progression-free survival in advanced ovarian cancer.

BACKGROUND: The nucleotide excision repair (NER) pathway is the principal DNA repair pathway for removing bulky platinum DNA adducts. Suboptimal DNA repair may lead to improved response to platinum agents. The objective of this study was to determine whether single-nucleotide polymorphisms (SNPs) in NER pathway genes could be markers of platinum response in ovarian cancer. METHODS: The authors identified patients with advanced-stage, papillary serous ovarian cancer who underwent primary cytoreductive surgery followed by platinum-based chemotherapy. DNA was isolated from peripheral blood specimens. Twenty-two SNPs within NER genes (xeroderma pigmentosum [XP] complementation group A [XPA], XPB/excision repair cross-complementing rodent repair deficiency, complementation group 3 [ERCC3], XPC, XPD/ERCC2, XPF/ERCC4, XPG/ERCC5, Cockayne syndrome group B protein [CSB]/ERCC8, ERCC1) were genotyped using polymerase chain reaction analysis. RESULTS: In total, 139 patients with stage III and IV papillary serous ovarian cancer were genotyped. The XPC (reference SNP 3731108 [rs3731108]) adenosine-guanine (AG)/AA genotype versus the GG genotype was associated with prolonged a progression-free survival (PFS) of 21.3 months versus 13.4 months (hazard ratio [HR], 0.63; 95% confidence interval [CI], 0.42-0.95; P = .03). The XPC (rs1124303) guanosine-thymidine (GT)/GG genotype versus the TT genotype was associated with a prolonged PFS of 22.8 months versus 14.9 months (HR, 0.47; 95% CI, 0.24-0.94; P = .03). The XPC poly(AT) (PAT) (-/+)/(-/-) genotype versus the (+/+) genotype was associated with a prolonged PFS of 17 months versus 11.6 months (HR, 0.56; 95% CI, 0.36-0.89; P = .01). The XPF/ERCC4 (rs12926685) cytidine-thymidine (CT)/CC genotype versus the TT genotype was associated with a prolonged PFS of 16.7 months versus 12.4 months (HR, 0.63; 95% CI, 0.41-0.95; P = .03). On multivariate analysis adjusting for breast cancer (BRCA) gene and cytoreductive surgery status, the XPC SNPs remained significantly associated with prolonged PFS. CONCLUSIONS: The current results indicated that XPC is a key component of the NER pathway that participates in DNA damage repair. SNPs in the XPC gene may represent novel markers of ovarian cancer response to platinum-based chemotherapy.

A mechanistic study of tumor-targeted corrole toxicity.

HerGa is a self-assembled tumor-targeted particle that bears both tumor detection and elimination activities in a single, two-component complex (Agadjanian et al. Proc. Natl. Acad. Sci. U.S.A.2009, 106, 6105-6110). Given its multifunctionality, HerGa (composed of the fluorescent cytotoxic corrole macrocycle, S2Ga, noncovalently bound to the tumor-targeted cell penetration protein, HerPBK10) has the potential for high clinical impact, but its mechanism of cell killing remains to be elucidated, and hence is the focus of the present study. Here we show that HerGa requires HerPBK10-mediated cell entry to induce toxicity. HerGa (but not HerPBK10 or S2Ga alone) induced mitochondrial membrane potential disruption and superoxide elevation, which were both prevented by endosomolytic-deficient mutants, indicating that cytosolic exposure is necessary for corrole-mediated cell death. A novel property discovered here is that corrole fluorescence lifetime acts as a pH indicator, broadcasting the intracellular microenvironmental pH during uptake in live cells. This feature in combination with two-photon imaging shows that HerGa undergoes early endosome escape during uptake, avoiding compartments of pH < 6.5. Cytoskeletal disruption accompanied HerGa-mediated mitochondrial changes whereas oxygen scavenging reduced both events. Paclitaxel treatment indicated that HerGa uptake requires dynamic microtubules. Unexpectedly, low pH is insufficient to induce release of the corrole from HerPBK10. Altogether, these studies identify a mechanistic pathway in which early endosomal escape enables HerGa-induced superoxide generation leading to cytoskeletal and mitochondrial damage, thus triggering downstream cell death.

Tumor detection and elimination by a targeted gallium corrole.

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-targeted corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. Targeted delivery in vivo leads to tumor cell death while normal tissue is spared. These findings contrast with the effects of doxorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The targeted complex ablated tumors at >5 times a lower dose than untargeted systemic doxorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and targeting advantages over standard chemotherapeutic agents.

Corroles that bind with high affinity to both apo and holo transferrin.

The interactions of transferrin (Tf) with the water soluble corrole 1 and with its gallium (1-Ga) and manganese (1-Mn) complexes were studied to establish the possible utilization of corrole-transferrin conjugates for targeting these corroles to cells that express the transferrin receptor. The protein, in both its iron-free apo form (apoTf) and the iron-bound holo form (holoTf), was found to spontaneously bind all three derivatives. This conclusion was reached from titrations followed by several spectroscopic methods and dilution experiments measured by fluorescence. The such elucidated very small dissociation constant of 2 x 10(-7) M and 3 x 10(-8) M for 1-Ga with apoTf and holoTf, respectively and <10(-9) M for 1 with both protein forms are clearly relevant for the physiological concentration of transferrin in serum.

Large Field of View Scanning Fluorescence Lifetime Imaging System for Multimode Optical Imaging of Small Animals.

We describe a scanning fluorescence lifetime imaging (SFLIM) system that provides a large field of view (LFOV), using a femtosecond (fs) pulsed laser, for multi-mode optical imaging of small animals. Fluorescence lifetime imaging (FLIM) can be a useful optical method to distinguish between fluorophores inside small animals. However, difficulty arises when LFOV is required in FLIM using a fs pulsed laser for the excitation of the fluorophores at low wavelengths (<500nm), primarily because the field of view of the pulsed blue excitation light generated from the second harmonic of the fs pulsed light is limited to about a centimeter in diameter due to the severe scattering and absorption of the light inside tissues. Here, we choose a scanning method in order to acquire a FLIM image with LFOV as one alternative. In the SFLIM system, we used a conventional cooled CCD camera coupled to an ultra-fast time-gated intensifier, a tunable femtosecond laser for the excitation of fluorophores, and an x-y moving stage for scanning. Images acquired through scanning were combined into a single image and then this reconstructed image was compared with images obtained by spectral imaging. The resulting SFLIM system is promising as an alternative method for the FLIM imaging of small animals, containing fluorophores exited by blue light, for LFOV applications such as whole animal imaging.

Effective high-capacity gutless adenoviral vectors mediate transgene expression in human glioma cells.

Glioblastoma multiforme (GBM) is the most common subtype of primary malignant brain tumor. Although serotype 5 adenoviral vectors (Ads) have been used successfully in clinical trials for GBM, the capacity of Ads to infect human glioma cells and the expression of adenoviral receptors in GBM cells have been challenged. In this report, we studied the expression of three molecules that have been shown to mediate adenoviral entry into cells, i.e., coxsackie and adenovirus receptor (CAR), integrin alphavbeta3 (INT), and major histocompatibility complex class I (MHCI), in rodent glioma cell lines and low-passage primary cultures and cell lines from human GBM. We correlated levels of expression of CAR, INT, and MHCI with transduction efficiency elicited by several high-capacity helper-dependent adenoviral vectors (HC-Ads). Expression levels of adenoviral receptors were variable among the different GBM cells studied. HC-Ad-mediated therapeutic gene expression was efficient, ranging between 20 and 80% of the total target cells expressing the encoded transgenes. Our results show no correlation between the levels of CAR, INT, or MHCI molecules and the levels of transgene expression or the number of GBM cells transduced. We conclude that expression levels of adenoviral receptors do not predict their transduction efficiency or biological function.

Specific delivery of corroles to cells via noncovalent conjugates with viral proteins.

PURPOSE: Corroles are amphiphilic macrocycles that can bind and transport metal ions, and thus may be toxic to cells. We predicted that anionic corroles would poorly enter cells due to the negatively charged cell membrane, but could be ideal tumor-targeted drugs if appropriate carriers enabled delivery into tumor cells. In this work, we test the hypothesis that recombinant cell penetrating proteins of the adenovirus (Ad) capsid form noncovalent conjugates with corroles to facilitate target-specific delivery and cell death. METHODS: Corroles mixed with recombinant proteins were tested for conjugate assembly, cell penetration, stability, targeted binding, and cell killing in vitro. RESULTS: Sulfonated corroles entered cells only with carrier proteins, and formed stable complexes with recombinant Ad capsid proteins. ErbB receptor-targeted conjugates were cytotoxic to ErbB2-positive but not ErbB2-negative breast cancer cells, whereas molar equivalents of free corrole had no effect on these cells. CONCLUSIONS: Sulfonated corroles are cytotoxic to ErbB2-positive breast cancer cells when delivered by a targeted cell penetrating protein. The relatively low dose required to accomplish this compared to untargeted compounds suggests that corroles may lend themselves to targeted therapy. Importantly, the amphiphilicity of corroles enables a unique approach to bioconjugate formation whereby the carrier and drug form a stable complex by noncovalent assembly.

Regulatable gutless adenovirus vectors sustain inducible transgene expression in the brain in the presence of an immune response against adenoviruses.

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.