Utilization of a cell-penetrating peptide-adaptor for delivery of HPV protein E2 into cervical cancer cells to arrest cell growth and promote cell death

Cervical cancer is the second leading cause of cancer deaths in women worldwide. Human papillomavirus (HPV) is the causative agent of nearly all forms of cervical cancer, which arises upon viral integration into the host genome and concurrent loss of regulatory gene E2. E2 protein regulates viral oncoproteins E6 and E7. Loss of E2 upon viral integration results in unregulated expression and activity of E6 and E7, which promotes carcinogenesis. Previous studies using gene-based delivery show that reintroduction of E2 into cervical cancer cell lines can reduce proliferative capacity and promote apoptosis. However, owing in part to limitations on transfection in vivo, E2 reintroduction has yet to achieve therapeutic usefulness. A promising new approach is protein-based delivery systems utilizing cell-penetrating peptides (CPPs). CPPs readily traverse the plasma membrane and are able to carry with them biomolecular ‘cargos’ to which they are attached. Though more than two decades of research have been dedicated to their development for delivery of biomolecular therapeutics, the full potential of CPPs has yet to be realized as the field is hindered by the tendency of CPP-linked cargos to be trapped in endosomes as well as having significant off-target potential in vivo. Using a CPP-adaptor system that reversibly binds cargo thereby overcoming the endosomal entrapment that hampers other CPP methods, bioactive E2 protein was delivered into living cervical cancer cells, resulting in inhibition of cellular proliferation and promotion of cell death in a time- and dose-dependent manner. The results suggest that this nucleic acid- and virus-free delivery method could be harnessed to develop novel, effective protein therapeutics for treatment of cervical cancer.


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Human papillomavirus is a sexually transmitted virus and the causative agent of multiple 45 forms of cancer including cervical, vaginal, oropharyngeal, anal, penile and vulvar. Vaccines 46 against the most common cancer-causing strains of HPV have been approved in the U.S. However, cervical cancer remains the second leading cause of cancer-related death in women worldwide [1].
CBS-E2 cargos were labeled with DyLight 550 (ThermoFisher, USA), incubated with or without 157 unlabeled TAT-CaM in equimolar amounts and brought to a total volume of 50 µL in binding 158 buffer. Complexes were then added to 200 µL plain glucose-free media and introduced to cells at 159 a final concentration of 1 uM. Uptake was performed in a humidified incubator at 37°C with 5% 160 CO 2 injection for 1 hour with periodic rocking (every 15 min) to ensure even distribution. After 1 161 hour, media were removed and cells were washed 5 times with calcium-containing phosphate 162 buffered saline (PBS with 1mM CaCl 2 ). Next, cells were counterstained with 2 µM CellTracker 163 Green (5-chloromethylfluorescein diacetate dye) (ThermoFisher) followed by NucBlue 164 (ThermoFisher) per manufacturer's protocols to stain the cytoplasmic and nuclear compartments 165 of the cells. After staining, cells were washed 3x with calcium-containing PBS and complete cell 166 culture media was added to each well. Cells were imaged using a 40x EC Plan-Neofluar objective 167 with a numerical aperture value of 1.3. Image analysis was performed as previously described 168 [27].

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(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)  CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay). In the same population of cells, cell 173 death was assayed by release of LDH into cell culture media (Promega CytoTox 96 ® Non-174 Radioactive Cytotoxicity Assay). Cells were seeded into 96-well plates at either 2.5 x10 3 or 2.5 175 x10 4 in 100 µL of phenol-red free cell culture media and allowed to adhere to the plate overnight   overcoming the 'endosomal escape' problem that has plagued the field [26]. In this study an E2 204 construct from HPV-16 that contained an N-terminal CBS tag was expressed and purified.

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That the kinetic constants were highly similar to other cargos examined previously [27,28] 210 validated the utility the approach for delivery of E2. TAT-CaM was used to deliver free bioactive E2 protein into the human HPV-16+ cervical 222 cancer cell line SiHa. Given significant artifacts resulting from fixation that have confounded delivery of fluorescently labeled CBS-E2 by TAT-CaM (Fig 2). To verify that TAT-CaM mediated distinctive pattern of CBS-E2 redistribution to regions structurally similar to spindle fiber 249 formations (white arrows; Fig 2C-F). Circular clusters of E2 formed on DNA were observed at the 250 onset of mitosis (white arrows; Fig 2C, D), suggestive of localization to aster microtubules as 251 previously described [30,32]. In cells undergoing anaphase and telophase, CBS-E2 clustered on 252 the midplane (white arrows; Fig 2E, F). These data demonstrate that CBS-E2 was delivered in 253 bioactive form and that the CBS tag did not significantly affect activity. groups showed similar growth rates while cells treated with 4 µM E2 failed to proliferate (Fig 3B).

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Microscopic analysis of cells on day 4 further corroborated these findings (Fig 3C-E). Untreated 267 and TAT-CaM treated cells exhibited normal morphology while E2 treated cells overwhelming 268 became flattened out, with a loss in typical spindle-like morphology, and exhibited intracellular 269 stress granule-like formations (Fig 3E). Collectively, these data support that 3 doses of E2 protein over a three-day time-period are sufficient to significantly reduce cellular proliferation within this 271 population of cells.  (Fig 3B). Microscopic analysis supported these findings (Fig 3 F-

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H). In untreated and TAT-CaM treated groups, cells became over-confluent and crowded the wells 289 (Fig 3F, G). E2-treated cells showed no visual evidence of doubling, however, some cells within 290 the population regained normal spindle-like morphology (Fig 3H). We hypothesize that these cells 291 might represent a subpopulation of harder to treat 'persister' cells. To test for this, cells were 292 collected and re-seeded at equal density. After 7 days in culture, cells were collected and counted ( Fig 3I). E2 treated cells regained normal growth kinetics ( Fig 3I) and normal morphology (data 294 not shown) indistinguishable from untreated or TAT-CaM treated cells. These data suggest that at 295 the cell-to-peptide ratios employed, a sub-population of cells underwent senescence while others 296 were seemingly unaffected or more resistant to E2's effects.

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To test the effect of cell-to-peptide ratios a dose-response assay was performed using the 298 same protocol with the exception that the starting cell number was lowered 10-fold. While 0.1 µM 299 doses had little effect, cells showed a dramatic reduction in metabolic activity at only 1 µM (75% 300 loss; Fig 4A). Similar observations were made with 10 µM doses, suggesting that at doses >1 µM 301 there is a 'plateau effect,' in that higher doses had no discernible effect (Fig 4A). Within the same 302 population of cell, cell death was tested each day by measuring total LDH levels in the media.

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Results showed significantly high levels of LDH in all E2 treatment groups (Fig 4B). The much 304 smaller level of LDH activity in controls was attributed to retention of normal growth rates leading 305 to overconfluency. Next, E2's ability to induce cell death in a non-cervical cancer human 306 microvascular endothelial cell line (HMEC) was tested. The same LDH leakage assay was 307 performed as above using the highest dose group of TAT:CaM & CBS-E2 (10 µM) in both SiHa 308 and HMEC cell lines. SiHas showed significantly higher levels of cell death when compared with 309 all other treatment groups while there was no discernable effect on HMECs following E2 delivery 310 (Fig 5A). Microscopic analysis of cells on day 3 qualitatively corroborated these results (Fig 5 B-

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I). Collectively, these data support the hypothesis that direct delivery of E2 protein into living 312 cervical cancer cells can inhibit cellular proliferation or induce cell death and, further, suggest that 313 these differential outcomes may be a function of dose. That E2 did not induce cell death in the This work sought to address the hypothesis that bioactive viral E2 protein could be 336 delivered directly into living cervical cancer cells via the TAT-CaM CPP-adaptor system to inhibit 337 cellular proliferation and/or promote cell death. CBS-E2 showed the expected high affinity, calcium-dependent binding kinetics with TAT-CaM (Fig 1), well within the range of constants may be favored while at high ratios cell death is (Figs 3 and 4)