Last year, more than 70,000 people in the United States were diagnosed with head and neck cancers, and its rates have steadily risen worldwide. These cancers arise from squamous epithelial cells lining these areas, with tobacco and alcohol as the primary culprits. However, for about 30 percent of patients, human papillomavirus (HPV) is to blame.
While researchers have studied HPV-positive cancer cells in advanced stages, the mechanisms driving the earliest events in the transformation of healthy cells into malignant ones remain unclear. This motivated molecular biologist Jorge Silvio Gutkind at the University of California, San Diego to capture the elusive but crucial factors that cause healthy stem cells to infiltrate tissue as they transition to cancer cells.
While certain HPV proteins are known to promote cancer, they don’t act alone. Gutkind and his colleagues previously found that alterations in the Hippo pathway, which regulates tissue growth and organ size, also contributed to head and neck squamous cell carcinomas.1
Jorge Silvio Gutkind studies growth-promoting signal transduction pathways in cancer to better understand cancer progression.
Kyles Dykes/University of California San Diego Health Sciences
A disruption in the Hippo pathway resulted in the activation of yes-associated protein (YAP), a transcription factor involved in stem cell maintenance and growth. Because of this, the researchers were interested in YAP’s role as a cancer driver. Their new study, published in Nature Communications, revealed that unrestrained YAP expression, in combination with HPV oncoproteins, triggered a rapid cascade of genetic and cellular changes that reprogrammed normal stem cells into cancerous ones.2 These findings illuminate the earliest steps of cancer initiation and highlight potential targets for detecting and preventing HPV-positive cancers.
Gutkind and his team focused on HPV-positive oral cancer, which involves the loss of function of two key tumor suppressor genes, tumor protein 53 and cyclin-dependent kinase inhibitor 2A. The researchers can model this either by editing the genes themselves or inhibiting their expression with HPV E6 and E7 oncoproteins. These two oncoproteins help drive HPV-associated cancers by activating the Hippo pathway to promote cell proliferation. Although YAP is a known effector downstream of Hippo, Gutkind aimed to characterize its direct effects alongside E6-E7 expression on cancer initiation.
The researchers used a genetic mouse model that conditionally expressed either constitutively active YAP (Y), E6-E7 (E), or a combination of YAP and E6-E7 transgenes (EY). Feeding the mice doxycycline-infused chow activated these genes, allowing the researchers to observe their combined effects on tumor development.
Gutkind was surprised by how quickly the tumors formed. “Within no more than two weeks, we [saw] the cancer,” he remarked. EY mice developed thickened tongues with deeply invasive macroscopic lesions, while those with only E or Y genes had fewer and smaller lesions. Control mice lacking these genetic alterations developed no lesions.
“Their comparison of the activation of E6 and E7 to the hyperactivation of YAP is an interesting one that I haven’t seen done directly in that way,” said Elizabeth White, a molecular biologist at Tufts University who was not involved in the study.
To pinpoint the genes linked to tumor initiation, the team performed bulk RNA sequencing (RNAseq) on mouse tongues 15 days after gene induction. E mice expressed 60 unique genes; Y mice exhibited 167 differentially expressed genes compared to control mice. However, EY mice expressed more than 2,000 unique genes, which were related to cell proliferation, epithelial cell development and identity, and inflammatory responses.
Compared to normal cells, EY cells also exhibited increased epigenetic reprogramming, resulting in increased chromatin accessibility of numerous genes, which promoted proliferation, invasion, and inflammation. Notably, the team saw an association with the mammalian target of rapamycin (mTOR) signaling pathway, commonly activated in cancer.
Then, Gutkind aimed to track the progression of oral stem cell fate over time, focusing on cellular diversity to better understand the subtle changes that drive the transition from healthy stem cells to a cancerous state. To examine this cellular heterogeneity in more detail, the researchers used single-cell RNAseq. “At the single-cell level, we can [see] every individual cell and see what decisions they make,” Gutkind remarked. The team analyzed live cells from the control mice and all three types of transgenic mice and identified eight distinct cell clusters: five corresponding to normal renewing epithelial stem cells and three associated with the transgene-expressing groups (E, Y, and EY). Among these, EY cells formed a cluster resembling tumor-initiating (TI) cells. These formerly normal stem cells began proliferating and acquiring invasive properties—a hallmark of cancer.
Next, the researchers used transcriptomics and microscopy to explore the processes by which these cells initiated tissue invasion. To their surprise, they found that TI cells recruited myeloid immune cells to the tumor microenvironment.
The team assessed TI gene expression and found increased markers specific to recruiting granulocytes. These granulocyes can produce collagenase, an enzyme that breaks down collagen in tissue. Tissue imaging confirmed this, showing fewer collagen fibrils in EY tissue compared to normal tissue. Flow cytometry provided further evidence showing an increase in infiltrating immune cells. The researchers believe these immune cells and TI cells work together to degrade collagen, clearing a path for tumor invasion. Gutkind also remarked that co-opting immune cells may be an early strategy for cancer cells to evade immune detection.
“We can also identify, by also focusing on the immune cells and other cells, all of the interplay [and communication] between the cells to initiate the cancer,” added Gutkind. “That was very exciting.”
Overall, the researchers demonstrated that YAP activation affected cell programs in oral epithelial progenitor cells, which drove cells towards more aberrant proliferation and led to the recruitment of myeloid cells to aid tissue infiltration.
For Gutkind, the next step is to apply the same technology to understand the progression in HPV-negative oral cancers and develop YAP inhibitors. White agreed and noted that, “There's great evidence that [YAP is] a very powerful cancer driver, so there’s a great opportunity for therapeutic intervention in that sense.”
- Faraji F, et al. Genomic Hippo pathway alterations and persistent YAP/TAZ activation: New hallmarks in head and neck cancer. Cells. 2022;11(8):1370.
- Faraji F, et al. YAP-driven malignant reprogramming of oral epithelial stem cells at single cell resolution. Nat Commun. 2025;16(1):498.
