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Article

Targeting STAT3 Promotes Tumor Cell Death and Enhances T-Cell Activity in HPV16-Positive Cancer

by
Ruben Prins
1,
Daniel J. Fernandez
1,
Diane M. Da Silva
2,
James Turkson
3,
De-Chen Lin
4 and
W. Martin Kast
1,2,4,*
1
Department of Immunology & Immune Therapeutics, USC/Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA
2
Department of Obstetrics & Gynecology, USC/Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA
3
Division of Medical Oncology, Department of Medicine at Cedars-Sinai, Los Angeles, CA 90048, USA
4
Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA
*
Author to whom correspondence should be addressed.
Cancers 2026, 18(4), 599; https://doi.org/10.3390/cancers18040599
Submission received: 15 January 2026 / Revised: 3 February 2026 / Accepted: 7 February 2026 / Published: 12 February 2026
(This article belongs to the Section Infectious Agents and Cancer)
Browse Figures
Versions Notes

Simple Summary

Standard treatment for human papillomavirus positive (HPV+) cancer includes surgical resection combined with chemo and/or radiation therapy. These can impair fertility, speech, or quality of life. This highlights the immediate need for better and less invasive treatment of HPV-driven cancers. The two viral proteins that put patients with an HPV infection at risk for cancer are able to suppress the immune-mediated clearance of HPV+ cells. Here we describe the efficacy of CPA-7, a drug that shuts down the STAT3 signaling pathway partially responsible for this immune suppression in HPV-driven tumors. This drug rescued the HPV-specific immune response in mice bearing HPV+ tumors and directly killed the cancer cells. Combined, these effects empowered CPA-7 to cure a significant percentage of mice bearing late-stage HPV+ tumors, highlighting the therapeutic efficacy of targeting STAT3 in HPV-driven disease.

Abstract

Background/Objectives: Human papillomavirus (HPV) oncoproteins early (E)6 and E7 cause upregulation of the IL-6 and IL-23 cytokines in HPV16+ cancers, contributing to tumor progression through enhanced tumor cell proliferation and suppression of the tumor specific adaptive CD8 T-cell response. The IL-6 and IL-23 receptors signal through signal transducer and activator of transcription 3 (STAT3) in the tumor microenvironment. Methods: To better understand how HPV-induced STAT3 signaling contributes to tumor progression and explore its therapeutic potential, we used the platinum (IV) compound CPA-7, a specific STAT3 inhibitor. CPA-7 was tested in vitro for its ability to inhibit STAT3 signaling, alter proliferation, and cause cell death in HPV16+ C3.43 tumor cells. In vivo, CPA-7 was tested for its ability to affect the HPV specific T-cell response, tumor growth, and survival in C3.43 tumor bearing mice. Results: In vitro, CPA-7 inhibited STAT3 signaling, reduced proliferation, and caused significant cell death to HPV16+ C3.43 cells. In vivo, CPA-7 eradicated early-stage HPV16+ tumors, while therapeutic treatment of late-stage tumors led to a systemically increased presence of tumor-specific CD8 T-cells and halted tumor progression. Conclusions: These results suggest that targeting STAT3 signaling downregulates tumor cell proliferation and induces tumor cell death. In addition, targeting STAT3 increases the HPV-specific anti-tumor adaptive immune response. Combined, this results in significantly reduced late-stage HPV16+ tumor progression.

Graphical Abstract

1. Introduction

Human papillomavirus (HPV) is the primary cause of cervical cancer and a subset of oropharyngeal cancers [1,2,3,4,5]. HPV has over 200 subtypes found in humans [6]. Based on tissue tropism, these viruses are divided into cutaneous (low risk, wart-causing) and mucosal HPV types. Mucosal HPVs are further classified as either low risk or high risk (hr) depending on the carcinogenic potential. Fifteen hr-HPVs have been identified as the probable cause for cancers in both the anogenital and head and neck regions, with HPV16 accounting for 50–90% of HPV+ cancer cases depending on the anatomical origin [1,7,8,9,10,11].
HPV16 early (E)6 and E7 oncoproteins are regarded as the catalysts for HPV-induced squamous intraepithelial lesions and subsequent progression to cancer. E6 expression causes degradation of P53, disrupting the ability of cells to detect mutations, while E7 manipulates retinoblastoma protein (pRB) and other components of the centrosome cycle to upregulate cell division [12,13,14]. In addition to impairing the cellular surveillance systems that maintain genomic integrity and promoting cell cycle progression, these HPV16 oncoproteins cause significantly enhanced production of IL-23 by macrophages in HPV16+ tumors [15,16]. IL-23 signals through the IL-23 receptor (IL-23R) on HPV16-specific CD8+ T cells and reduces their cytotoxicity and proliferative capacity, while IL-23R signaling in CD4+ T regulatory cells (Treg) increases their suppressive nature [17,18]. Our previous research suggests that this is indirectly caused by HPV16 oncoproteins, as their presence in the HPV16+ tumor microenvironment (TME) upregulates both the number of IL-23-expressing M2 macrophages and upregulates their individual IL-23 expression level through upregulating the transcription factor KLF2 [15].
IL-23, a dimer of IL12p40 and IL23p19, triggers IL-23R in immune cells, which causes the phosphorylation and activation of signal transducer and activator of transcription 3 (STAT3). It does so through activating STAT3 in three different ways. Signaling through the IL-23R intracellular domain not only activates JAK2, a canonical activator of STAT3, but the receptor also has two binding sites that activate the STAT3 src homology 2 (SH2) domain directly or through the PI3K/Akt and MAPK pathways [19]. STAT3 activation involves the phosphorylation of Tyrosine 705 located in the SH2 domain, which cause the dimerization of two STAT3 monomers, which subsequently translocate from the cytoplasm into the nucleus where they function as a transcription factor known to drive multiple hallmarks of cancer in the HPV16+ TME [20,21]. Similar to what is observed for IL-23, overall p-STAT3 levels in local tissue increase as HPV-driven disease progresses from low-grade lesions into cancer [22]. However, since IL-23 mainly signals in immune cells, the activation of STAT3 in cancer cells is more likely to occur through IL-6, which is upregulated by cancer cells directly through the presence of HPV16 E6 [23]. IL-6 has been implicated as a key player in the induction of IL-23 by antigen-presenting cells in HPV16+ cancers [16]. We therefore sought to explore the effects of increased STAT3 signaling on HPV16+ cancers and the tumor specific T cell immune response.
The platinum (IV) derived small-molecule-inhibitor CPA-7 targets the DNA binding domain of the activated STAT3 dimer and reduces the level of pSTAT3, thereby preventing STAT3 from functioning as a transcription factor [24]. Of the platinum (IV)-derived small molecules, CPA-7 is the only one that has been shown to have bioavailability and efficacy in treating syngeneic tumors in vivo [25,26]. Consistent with its mechanism of targeting constitutively active STAT3, CPA-7 exerts little effect on cell viability, proliferation, or apoptosis in cells without high basal STAT3 activity, such as human MDA-MB-453 breast cancer cells and NIH3T3 mouse fibroblasts [24]. Because HPV+ cancers drive STAT3 signaling in the TME through IL-6 and IL-23, our current study is aimed at understanding how STAT3 affects HPV16+ tumor cells and tumor infiltrating T lymphocytes and explores the efficacy of CPA-7 as a therapeutic compound in late-stage HPV16+ cancer.

2. Methods

2.1. Cell Culture

C3.43 cells [27,28], which are C57Bl/6 mouse embryonic cells (B6MECs) transformed with the full length HPV16 genome, were cultured in complete media containing Iscove’s modified DMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% FBS (Omega Scientific, Tarzana, CA, USA), 1X beta Mercaptoethanol, and 1X gentamycin (Thermo Fisher Scientific). Cells were seeded in triplicate in a 96-well tissue-culture-treated plate and incubated with indicated concentrations of STAT3 inhibitor CPA-7 for either 24 or 48 h. CPA-7 was synthesized in the lab of Dr. James Turkson, University of Hawaii, as previously described [24]. Proliferation was measured using CyQuant (Thermo Fisher Scientific) following manufacturer protocols. Briefly, 2000 cells were allowed to adhere for 3 h prior to addition of CPA-7. Cells were then incubated for indicated times, while 4 h prior to harvest, cells were seeded in separate wells at increasing density to create a standard curve. Media was washed out with PBS prior to addition of lysis buffer and staining reagent. Fluorescence was measured using a CLARIOstar plate reader.

2.2. Tumor Challenge

C3.43 cells were passaged twice and grown to 80% confluency prior to harvesting by using trypsin. Cells were then washed twice using 50 mL HBSS. Cells were counted and used if their viability exceeded 95%. C57BL/6 female mice (5–7 weeks) were shaved on the right flank and injected sc with 1 × 105 C3.43 in 100 μL of HBSS. CPA-7 was dosed at 5 mg/kg (i.p.) in 50 μL twice every week starting on indicated days and maintained for 10 treatments total. Depleting antibodies either for CD4, CD8 (Bio X Cell, Lebanon, NH, USA), or CD4 and CD8 (kindly provided by Dr. Alan Epstein, University of Southern California) were dosed at 400 ug two consecutive days and subsequent maintenance doses at 200 ug 2 times/week every week for the duration of the experiment. Prior to initiation of CPA-7 treatment, depletions were confirmed in the blood as described under flow cytometry. Tumor growth (LxWxD) was monitored until mice reached their humane endpoint (>1500 mm3, ulceration >50% of surface, body score drop). All animal experiments were approved under IACUC protocol 20065 (approved on 19 March 2025).

2.3. Flow Cytometry

Cell death and apoptosis was measured in trypsinized cells after incubation with CPA-7 or vehicle control using a propidium iodine and Annexin V (FITC) stain. These cells were then analyzed by flow cytometry using the FC500 (Beckman Coulter, Indianapolis, IN, USA).
Depletion of indicated cell populations in vivo was done through analyzing PBMCs obtained by retro-orbital bleeds through flow cytometry. Collected blood was treated with heparin to prevent clotting and spun down to remove plasma. ACK lysis buffer (Thermo Fisher Scientific) was used to lyse red blood cells, prior to washing with PBS containing 2% FBS (FACS). Cells were washed with PBS to remove cell debris prior to staining for Zombie Aqua (ZA, Biolegend, San Diego, CA, USA) at room temperature in the dark for 15 min. PBS was used to wash unbound ZA, and PBMC were then stained for CD45, CD3, CD4, and CD8 (Biolegend) for 20 min in FACS buffer in the dark on ice. Unbound antibody was washed off using FACS buffer prior to reading samples on BD FACSCanto II (BD, Franklin Lakes, NJ, USA). Fluorescence minus one was used to establish gating. Data was analyzed on FlowJo (Ashland, OR, USA).

2.4. Western Blot

C3.43 were cultured with CPA-7 (as described under cell culture) prior to harvesting in MPER (Thermo Fisher Scientific) containing a Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration in lysates was determined by a Bradford assay, where the lysate concentration was determined from a standard curve of BSA. A total of 100 ug of protein was then denatured with loading buffer at 96 °C for 10 min. Lysates were then loaded onto a bis-tris gel together with Seeblue plus II (Thermo Fisher Scientific) marker and run in MOPS buffer (Thermo Fisher Scientific). The iBlot system (Thermo Fisher Scientific) was used to transfer proteins from the gel onto a nitrocellulose membrane. The membrane was subsequently blocked using 5% nonfat dry milk and stained overnight on a rocker with primary antibodies against STAT3, (tyr705)p-STAT3, Cyclin D1, GAPDH, and beta actin (Cell Signaling Technologies, Danvers, MA, USA) overnight at 4 °C in the dark. The next day, the primary antibody was removed and the blot was washed three times with 10 mL PBS-T prior to incubation with a secondary antibody linked to either IRDye 800CW or IRDye 680RD (Li-COR, Lincoln, NE, USA) for 1 h at room temperature. Blots were washed three more times with PBS-T prior to imaging (Li-COR).

3. Results

3.1. CPA-7 Inhibits STAT3 Signaling, Causes a Reduction in Proliferation, and Increases Cell Death in HPV16+ C3.43 Tumor Cells

To investigate the role of STAT3 signaling in the progression of HPV+ cancer, we used the small molecule inhibitor CPA-7. This platinum-derived compound binds to the binding pocket of the pSTAT3 dimer, preventing its subsequent signaling activity. We first sought to validate the ability of CPA-7 to inhibit STAT3 activity in HPV16-induced C3.43 tumor cells. Upon successful inhibition of STAT3 signaling, CPA-7 would be expected to reduce downstream genes reliant upon STAT3 as a transcription factor. At the protein level, CPA-7 has been reported to reduce pSTAT3 [25], the activated state of STAT3. Analysis of the C3.43 cells treated with CPA-7 confirmed that levels of STAT3 and p-STAT3 (Tyr705) were both reduced in these cells (Figure 1A). CPA-7 treatment of the C3.43 cells also caused downregulation of Cyclin D1, which is under transcriptional control of STAT3 (Figure 1B).
Next, we sought to understand the impact of CPA-7 on the proliferation rate of C3.43 cells. Within 24 h, proliferation of the C3.43 cells was significantly reduced with exposure to 10 uM CPA-7 (Figure 1C), while longer incubation for up to 48 h led to reduced proliferation at concentrations as low as 0.1 uM CPA-7 (Figure 1D).
Because CPA-7 is known to cause apoptosis and higher concentrations of CPA-7 led to observable membrane blebbing (Figure 2A, Figure S1), the C3.43 cells were stained for the presence of annexin V (AV) on the outer leaflet of the cell membrane as a measure for early apoptosis and propidium iodine to determine cell death. CPA-7 increased the presence of AV on the outer leaflet of the membrane in a dose-dependent manner (Figure 2B, Figure S2) and caused a precipitous drop in viability, as marked by increased propidium iodine (PI) staining (Figure 2C). At 24 h, many of the cells positive for AV were not yet positive for PI (Figure 2D), indicating that inhibiting STAT3 in C3.43 cells initiates apoptosis.
Collectively, this confirms that CPA-7 can inhibit STAT3 signaling in HPV16+ C3.43 cells and cause increased tumor cell death and reduced tumor cell proliferation.

3.2. Treatment of Early-Stage HPV+ Cancer with CPA-7 Leads to Tumor Regression That Relies on an Adaptive Immune Response Involving CD4 and CD8 T-Cells

When treating C3.43 tumor bearing mice with CPA-7 starting on the day of tumor challenge, we found that this had a dramatic impact on tumor progression, eradicating their presence and confirming STAT3 plays a significant role in the progression of HPV-driven tumors (Figure 3A). The mice with eradicated tumors (9/10) were subsequently rechallenged on day 38 on the opposite flank to determine immune memory. Of the nine rechallenged mice, four remained tumor free (Figure 3B). This shows early treatment of HPV16+ C3.43 tumors with CPA-7-induced full tumor regression and led to immune memory.
IL-23 signaling plays a significant role in suppressing the HPV specific immune response by lowering the ability of CD8 T-cells to proliferate and kill their target cells [15]. IL-23 signals through the CD8 T-cell IL-23 receptor, which uses STAT3 for signal transduction. Additionally, CPA-7 efficacy in B16 melanoma has previously been shown to be primarily dependent on CD4 and CD8 T-cells. To understand whether the effects observed by CPA-7 are due to interference with the HPV specific T-cell immune response, CPA-7 treatment was initiated after the depletion of CD4 and CD8 T-cells using their depletion antibodies (Figure 4A,B). Although the tumors grew significantly slower with CPA-7 treatment, even in the absence of CD4 and CD8 T-cells, blocking STAT3 prevented these mice from fully clearing their tumors (Figure 4C), showing that the efficacy of STAT3 inhibition is at least partially driven by suppressing the HPV16 specific T-cell immune response.
To better understand the reliance of CPA-7 treatment on either CD4 or CD8 T-cells, subsets were depleted individually (Figure 5A,B). Surprisingly, tumors treated with individual depletion of CD4 and CD8 T-cells starting on the day of tumor challenge did not affect the CPA-7 efficacy to the same extent (Figure 5C). Only the CD8 T-cell-depleted mice challenged with the C3.43 cells continued to have palpable mass at the tumor site, which did not grow out after the CPA-7 treatment was completed (Figure 5D).
To uncover whether STAT3 inhibition can be used therapeutically and to better understand the dynamic of the adaptive immune response and treatment efficacy, CPA-7 treatment was initiated in early- (day 7) and late-stage (day 15) established C3.43 tumors. CPA-7 significantly inhibited tumor growth in the treatment of both early- and late-stage tumors (Figure 5E).

3.3. CPA-7 Successfully Halts Tumor Progression in Late-Stage HPV16+ Tumors and Induces a Systemic HPV16 Specific CD8 T-Cell Response

Because CPA-7 causes tumor regression in the treatment of early-stage tumors, even when depleting CD4 and CD8 T-cells, it is difficult to elucidate the effect of CPA-7 on these T-cell subsets and the role STAT3 signaling plays in their activity. Therefore, depletion was subsequently initiated on day 14 and CPA-7 treatment was delayed to start on day 20 with the intention to grow the overall tumor mass to a point where CPA-7 can no longer reliably eradicate the tumor. Despite treating more advanced and larger tumors, the tumor growth was significantly inhibited with treatment, although most tumors no longer fully regressed (Figure 6A). Only CD8-depleted mice showed an increased trend of tumor growth as compared with the CPA-7-depleted group. Additionally, none of the tumors fully regressed when CD8 T-cells were depleted, while a third of CPA-7-treated mice (3/9) were still able to fully clear their tumor burden.
To understand the effects of treatment on the CD4 and CD8 T-cell populations, blood was collected after 4 weeks of CPA-7 treatment. CPA-7 did not affect the overall levels of the CD4 (Figure 6B) or CD8 T-cells (Figure 6C), but both the CPA-7 and CPA-7 + CD4 T-cell depletion groups showed significantly elevated levels of HPV16-specific CD8 T-cells, as measured by HPV16 E7 tetramer staining (Figure 6D). Depletion of CD8 T-cells caused a trend of diminished CPA-7 efficacy (p = 0.075). The tumors in the CPA-7 + CD4 T-cell depletion group fully regressed and showed an increase in both overall CD8 T-cells and HPV16-specific CD8 T-cells, indicating that the effects of CPA-7 are not dependent on CD4 T-cells, but rather can function together with CD8 T-cells no longer inhibited by the presence of inhibitory CD4+ T-cells.

4. Discussion

HPV16 E6 upregulates IL-6 expression in cancer cells [23], which can reduce the ability of myeloid cells to mount an effective adaptive immune response in HPV16+ cancer treated with therapeutic vaccines [29]. In addition, mature human antigen presenting cells upregulate IL-23 in response to IL-6 [16], which, similar to IL-6, has been shown to suppress the efficacy of HPV therapeutic vaccination. Combining the upregulated expressions of both IL-6 and IL-23, there exists a significant pressure toward STAT3 signaling in HPV16+ tumors since receptors for these cytokines are found both on cancer cells and immune cells throughout the tumor microenvironment. Our data provides further evidence that STAT3 and its functions could be central to HPV-16-incuded cancers. Our data show that the inhibition of STAT3 activity occurs in both the tumor cells and immune cells and that the collective changes contribute to induction of the antitumor response observed in the HPV16+ C3.43 tumor model.
Early research linked elevated STAT3 activity to apoptosis resistance in cancer cells [30], and has since been associated with tumor cell proliferation; angiogenesis; inflammation; and other hallmarks of cancer, such as metastasis and invasion, for a variety of cancers [21]. Targeting STAT3 therapeutically is not a novel concept. Original approaches included phosphorylated proteins that would fit into the SH2 binding domain on STAT3 monomers, preventing them from reciprocally binding to another phosphorylated monomer [31]. This had great efficacy on cell free lysates, but its IC50 was greatly reduced when introduced into an in vivo cell system. Over time, the strategy of inhibiting the SH2 pocket by a small peptide has therefore shifted to synthetic small molecule inhibitors, STAT3 peptidomimetics, decoy DNA, and antisense oligonucleotides able to target the expression or function STAT3 proteins [24,32,33,34,35]. Although many have made it into clinical trials, STAT3 inhibitors have yet to receive FDA approval as a standalone treatment. Notable in this is the small molecule Napabucasin, which completed a randomized phase III trial regarding treatment of advanced colorectal cancer [36]. Although the improvement in overall survival (OS) did not reach statistical significance when looking at all patients, the treatment did significantly improve OS in those patients that were determined to overexpress activated pSTAT3.
CPA-7, a potent platinum (IV) based small molecule STAT3 inhibitor, has previously been shown as effective in treatment of murine prostate cancer [25], peripheral glioblastoma [37], melanoma [26], and bladder cancer [26] tumor models, where it significantly reduced tumor growth and elicited an antitumor immune response. We show for the first time the impact of CPA-7 on a HPV16+ tumor. CPA-7 significantly reduces levels of p-STAT3 (Figure 2A) and its downstream target Cyclin D1 in the HPV16+ C3.43 tumor cells after 48 h (Figure 2B), leading to a significant reduction in the rate of proliferation and viability (Figure 1C,D and Figure 2C). The observed reduction of total STAT3 protein in response to CPA-7 treatment is possibly caused by STAT3 autoregulating its own promoter [38,39]. These results help explain the observation in vivo, where CPA-7 is able to cause complete regression of early-stage tumors (Figure 3B) while significantly inhibiting the rate of tumor progression of late-stage cancer (Figure 5C and Figure 6A), in part through the effects that it exerts directly on the HPV16+ C3.43 cells. These findings emphasize that CPA-7 works better in HPV+ cancer than any other cancer studied, likely due to the virus-driven stimulation of STAT3 signaling.
Our recent work has suggested that increased levels of IL-23 cause suppression of the CD8 T-cell specific immune response in HPV16+ cancers, indicating that these T-cells incur increased IL-23R induced STAT3 signaling [15]. The potential role of sustained STAT3 signaling in suppressing the tumor specific immune response is further highlighted by findings that STAT3 signaling in the myeloid cells of the HPV+ cancer tumor environment can lower their ability to initiate an adaptive immune response. STAT3 antisense oligonucleotides fused with CpG are able to target elevated STAT3 in tumor associated dendritic cells and M2 macrophages, leading to increased CD8 T-cell recruitment in treatment of an HPV16+ head and neck squamous cell carcinoma tumor model [33]. This aligns with recent findings showing that STAT3 represses STAT5 activation in dendritic cells, thereby dampening their capacity to induce an immune response [40]. Likewise, STAT5 activation via IL-2 and GM-CSF receptors enhances CAR T-cell activation, degranulation, cytokine release, and cytotoxicity [41]. CPA-7-mediated STAT3 inhibition could therefore rebalance tumor immune microenvironment signaling from immunosuppressive STAT3 programs toward immunostimulatory STAT5 responses.
Treatment of the HPV16 E6/E7-expressing murine TC-1 tumor model has similarly shown induction of an HPV-specific immune response upon inhibition of STAT3 through S3I-201 [42]. However, none of these treatments led to tumor regression and rather showed significant reduction in the rate of tumor growth. Our work shows that STAT3 inhibition increases HPV16-specific CD8 T-cells systemically and is effective in treatment of HPV16+ C3.43 tumors, providing further evidence that STAT3 poses a promising therapeutic target in treatment of HPV+ cancer. We show that CPA-7, unlike other STAT3 inhibition modalities, results in the complete eradication of HPV+ tumors and that this is dependent on the presence of both CD4 and CD8 T-cells. The observation that CPA-7 is able to cause a significant reduction in the size of late-stage C3.43 tumors in the absence of CD8 T-cells, therefore further highlighting that the efficacy of CPA-7 depends on more than just the anti-tumor specific immune response.
Platinum (IV) drugs that carry chloro on their axial groups have a higher reduction potential as compared with those that carry carboxylato or hydroxo. Reduction of platinum (IV) complexes inside the cell can generate platinum (II) complexes like cisplatin [43]. CPA-7, which carries one chloro group, likely causes cisplatin complex formation inside the cell, leading to enhanced cytotoxicity in addition to inhibiting STAT3. Although other STAT3 inhibition strategies might provide cleaner targeting of STAT3 signaling without toxicities associated with platinum (IV) complexes, the increased cytotoxicity from CPA-7 could be aiding the release of tumor-specific antigens, such as HPV16 E7. This would allow for an adaptive immune response to be initiated, explaining why an increased frequency of HPV E7-specific CD8 T-cells is observed systemically in mice bearing C3.43 tumors treated with CPA-7 (Figure 6D).
It is important to further note is that STAT3 functions as a central signaling node integrating proliferative, survival, and immunoregulatory cues in both tumor and immune cells [44]. Therapeutic modulation of STAT3 is therefore expected to have broad, context-dependent effects beyond the pathway examined in this study. In addition to the intended impacts on tumor cell growth and adaptive antitumor immunity, treatment with CPA-7 could influence other immune cell subsets (e.g., NK cells [45,46]), cytokine networks (IL-6, IL-10, IL-23), and stress-response pathways that also signal through STAT3 [44]. For example, STAT3 inhibition has been associated with reduced tumor-promoting inflammation [33,44], but in other settings, STAT3 activity supports host defense [47], wound healing, and maintenance of normal tissue homeostasis [48], raising the possibility that its inhibition could impair tissue repair or susceptibility to infection. These considerations highlight the importance of dose, schedule, and tissue specificity when targeting STAT3 in vivo. In future studies that dissect cell-type-specific responses through single-cell transcriptomics and proteomics, determining the efficacy in male mice to encompass HPV+ head and neck cancer that arise primarily in men [49], as well as longer-term safety and infection-susceptibility readouts, will be essential to more precisely define the therapeutic window and safety profile of CPA-7.
When considering immunotherapy approaches to combat HPV16+ cancers, it is clear that mounting an immune response to HPV16 E6 and E7 alone is not sufficient to regress established tumors as the tumor microenvironment is suppressive to CD8 T-cells [50,51]. CPA-7 is able to cause systemic upregulation of HPV16-specific CD8 T-cells and, therefore, provides a promising avenue for combination therapy with therapeutic vaccination strategies. Depletion of CD4 T-cells in combination with CPA-7 led to complete tumor regression. In this group of mice, the overall CD8 T-cells and HPV16 specific CD8 T-cells significantly increased. This shows that not only do these CD4 T-cells aid in tumor progression and suppress CD8 T-cell expansion but also that the intact CD8 T-cells are able to function uninhibited in the presence of CPA-7.
Collectively, our data show that CPA-7 is able to cause HPV16+ cancer cell death while increasing HPV specific CD8 T-cell cytotoxicity, which, when combined, reduces tumor growth. This highlights the potential for inhibitors of the STAT3 signaling pathway to be used as chemical modulators of immune cell functions in combination with therapeutic vaccines and other immunotherapies in the treatment of HPV-driven tumors.

5. Conclusions

Inhibition of STAT3 through CPA-7 in late-stage HPV16+ tumors causes tumor cell death, induces a significant HPV specific T-cell immune response, and significantly improves the overall survival in tumor-bearing mice.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/cancers18040599/s1, Figure S1: Figure 2A Full Western Blot; Figure S2: Figure 2B Full Western Blot.

Author Contributions

Conceptualization, R.P., J.T., D.-C.L. and W.M.K.; Methodology, R.P., D.J.F. and D.M.D.S.; Formal Analysis, R.P. and W.M.K.; Investigation, R.P. and D.J.F.; Resources, J.T. and W.M.K.; Writing—Original Draft Preparation, R.P.; Writing—Review and Editing, D.J.F., D.M.D.S., J.T., D.-C.L. and W.M.K.; Visualization, R.P.; Supervision, W.M.K.; Project Administration, W.M.K.; Funding Acquisition, W.M.K. All authors have read and agreed to the published version of the manuscript.

Funding

W.M. Kast holds the Walter A. Richter Cancer Research Chair, and this research project was funded by his National Institutes of Health grant R01 CA074397. Financial contributions through gifts from RF Brennan, S Bloch, IY Khandros and the Norris Auxiliary are gratefully acknowledged.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of the University of Southern California protocol #20065.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available through inquiries that can be directed to the corresponding author.

Acknowledgments

We thank the USC Norris Comprehensive Cancer Center Beckman Center for Immune Monitoring supported by National Institutes of Health Grant P30 CA014089 for assistance with flow cytometry. The graphical abstract was created using BioRender. Prins, R. (2026), https://BioRender.com/4rt9yoh accessed on 6 February 2026.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Protein lysate of C3.43 cells treated with CPA-7 for 48 h was analyzed by Western blot and stained for (A) p-STAT3, STAT3, and GAPDH or (B) Cyclin D1 and b-Actin with CPA-7-concentration-matched vehicle controls of DMSO. (C) Cyquant proliferation assay determining cell number after 24 h and (D) 48 h incubation of C3.43 cells with CPA-7 or concentration matched vehicle controls of DMSO. Ctrl represents cells treated with a lethal DMSO 2% concentration. For pSTAT3, STAT3, and Cyclin D1, relative expression was determined against untreated control. All WB relative expressions were normalized against the indicated housekeeping protein. Differences between the treatment conditions and DMSO controls in the Cyquant assay was determined using a 2-way ANOVA. * p < 0.05; ** p < 0.01.
Figure 1. Protein lysate of C3.43 cells treated with CPA-7 for 48 h was analyzed by Western blot and stained for (A) p-STAT3, STAT3, and GAPDH or (B) Cyclin D1 and b-Actin with CPA-7-concentration-matched vehicle controls of DMSO. (C) Cyquant proliferation assay determining cell number after 24 h and (D) 48 h incubation of C3.43 cells with CPA-7 or concentration matched vehicle controls of DMSO. Ctrl represents cells treated with a lethal DMSO 2% concentration. For pSTAT3, STAT3, and Cyclin D1, relative expression was determined against untreated control. All WB relative expressions were normalized against the indicated housekeeping protein. Differences between the treatment conditions and DMSO controls in the Cyquant assay was determined using a 2-way ANOVA. * p < 0.05; ** p < 0.01.
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Figure 2. HPV16+ C3.43 cells were treated for 24 h with indicated concentrations of CPA-7. (A) brightfield images of C3.43 cells after 24 h incubation with CPA-7. (B) Annexin V, (C) PI, and (D) Annexin V+PI staining of C3.43 cells treated with CPA-7 for 24 h. Bars show mean +/- SEM. A 2-way ANOVA was used to determine significance of treatment effects. ** p < 0.01; **** p < 0.0001.
Figure 2. HPV16+ C3.43 cells were treated for 24 h with indicated concentrations of CPA-7. (A) brightfield images of C3.43 cells after 24 h incubation with CPA-7. (B) Annexin V, (C) PI, and (D) Annexin V+PI staining of C3.43 cells treated with CPA-7 for 24 h. Bars show mean +/- SEM. A 2-way ANOVA was used to determine significance of treatment effects. ** p < 0.01; **** p < 0.0001.
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Figure 3. On day 0, C57BL/6 mice were challenged with C3.43 tumors on the right flank and received either CPA-7 (20 mg/kg) or a DMSO vehicle control 2 times/week for 5 consecutive weeks. (A) The tumor growth rate and (B) survival were monitored. One week post-euthanasia of the control group, CPA-7 mice without tumors received a secondary C3.43 tumor challenge on the left flank on day 38. Effects of CPA-7 were confirmed in three independent experiments. The data represents one experiment with 10 mice/group. The data points represent the mean tumor volume ±SEM.
Figure 3. On day 0, C57BL/6 mice were challenged with C3.43 tumors on the right flank and received either CPA-7 (20 mg/kg) or a DMSO vehicle control 2 times/week for 5 consecutive weeks. (A) The tumor growth rate and (B) survival were monitored. One week post-euthanasia of the control group, CPA-7 mice without tumors received a secondary C3.43 tumor challenge on the left flank on day 38. Effects of CPA-7 were confirmed in three independent experiments. The data represents one experiment with 10 mice/group. The data points represent the mean tumor volume ±SEM.
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Figure 4. Blood from mice that were left (A) untreated or (B) treated with CD4- and CD8-depleting antibodies was collected and CD45+, ZA-, and CD3+ cells were analyzed for the presence of CD4 or CD8 T-cells by flow prior to C3.43 tumor challenge. CPA-7 (20 mg/kg) treatment was initiated on the day of the tumor challenge 2 times/week for 10 total treatments. CD4/CD8 antibodies were dosed 2 times/week for duration of experiment. (C) Individual tumor growth curves of mice in each respective treatment. The data represents one experiment with 10 mice/group. The data points represent individual tumor growth curves.
Figure 4. Blood from mice that were left (A) untreated or (B) treated with CD4- and CD8-depleting antibodies was collected and CD45+, ZA-, and CD3+ cells were analyzed for the presence of CD4 or CD8 T-cells by flow prior to C3.43 tumor challenge. CPA-7 (20 mg/kg) treatment was initiated on the day of the tumor challenge 2 times/week for 10 total treatments. CD4/CD8 antibodies were dosed 2 times/week for duration of experiment. (C) Individual tumor growth curves of mice in each respective treatment. The data represents one experiment with 10 mice/group. The data points represent individual tumor growth curves.
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Figure 5. Blood from mice that were (A) treated with CD4-depleting antibodies or (B) CD8-depleting antibodies was collected, and immune cells (CD45+, ZA−) were analyzed for presence of CD4 or CD8 T-cells (CD3+ and CD4 or CD8+) by flow prior to the C3.43 tumor challenge. (C) The mice received the indicated antibody treatments in combination with either CPA-7 or a vehicle control starting on day of tumor challenge. (D) Call out from panel C showing the average tumor sizes observed in the treated mice. (E) Mice bearing C3.43 tumors receiving CPA-7 treatment that was initiated on indicated days after the tumor challenge. The data represents one experiment with 10 mice/group. The data points represent the mean tumor volume ±SEM.
Figure 5. Blood from mice that were (A) treated with CD4-depleting antibodies or (B) CD8-depleting antibodies was collected, and immune cells (CD45+, ZA−) were analyzed for presence of CD4 or CD8 T-cells (CD3+ and CD4 or CD8+) by flow prior to the C3.43 tumor challenge. (C) The mice received the indicated antibody treatments in combination with either CPA-7 or a vehicle control starting on day of tumor challenge. (D) Call out from panel C showing the average tumor sizes observed in the treated mice. (E) Mice bearing C3.43 tumors receiving CPA-7 treatment that was initiated on indicated days after the tumor challenge. The data represents one experiment with 10 mice/group. The data points represent the mean tumor volume ±SEM.
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Figure 6. Mice bearing C3.43 tumors were therapeutically treated with depleting antibodies (isotype control, CD4, or CD8), starting on day 14, in combination with either CPA-7 or vehicle control, starting day 20 after depletions were confirmed. (A) Tumor growth was monitored and blood was drawn on day 40 to measure the levels of (B) CD4+, (C) CD8+, and (D) HPV E7 tetramer+ CD8 T-cells. The data represent one experiment with 9 mice/group. The data points represent mean tumor volume ± SEM. The line and bar graphs represent the mean ± SEM. A 2-way ANOVA was used to determine the differences between treatment groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 6. Mice bearing C3.43 tumors were therapeutically treated with depleting antibodies (isotype control, CD4, or CD8), starting on day 14, in combination with either CPA-7 or vehicle control, starting day 20 after depletions were confirmed. (A) Tumor growth was monitored and blood was drawn on day 40 to measure the levels of (B) CD4+, (C) CD8+, and (D) HPV E7 tetramer+ CD8 T-cells. The data represent one experiment with 9 mice/group. The data points represent mean tumor volume ± SEM. The line and bar graphs represent the mean ± SEM. A 2-way ANOVA was used to determine the differences between treatment groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
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MDPI and ACS Style

Prins, R.; Fernandez, D.J.; Da Silva, D.M.; Turkson, J.; Lin, D.-C.; Kast, W.M. Targeting STAT3 Promotes Tumor Cell Death and Enhances T-Cell Activity in HPV16-Positive Cancer. Cancers 2026, 18, 599. https://doi.org/10.3390/cancers18040599

AMA Style

Prins R, Fernandez DJ, Da Silva DM, Turkson J, Lin D-C, Kast WM. Targeting STAT3 Promotes Tumor Cell Death and Enhances T-Cell Activity in HPV16-Positive Cancer. Cancers. 2026; 18(4):599. https://doi.org/10.3390/cancers18040599

Chicago/Turabian Style

Prins, Ruben, Daniel J. Fernandez, Diane M. Da Silva, James Turkson, De-Chen Lin, and W. Martin Kast. 2026. "Targeting STAT3 Promotes Tumor Cell Death and Enhances T-Cell Activity in HPV16-Positive Cancer" Cancers 18, no. 4: 599. https://doi.org/10.3390/cancers18040599

APA Style

Prins, R., Fernandez, D. J., Da Silva, D. M., Turkson, J., Lin, D.-C., & Kast, W. M. (2026). Targeting STAT3 Promotes Tumor Cell Death and Enhances T-Cell Activity in HPV16-Positive Cancer. Cancers, 18(4), 599. https://doi.org/10.3390/cancers18040599

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