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Page 8 of 12 David. J Cancer Metastasis Treat 2022;8:32 https://dx.doi.org/10.20517/2394-4722.2022.71

Classical PDA cells are highly secretory, producing not only mucins but also large quantities of gastrokines
such as GKN2, GKN3, TFF1, and TFF3 [31,58] . Mucin and gastrokine production are also associated with
PanIN lesions [3,59,60] , an observation that suggests classical PDA and PanIN are closely related entities. The
[61]
high-level production of secreted proteins in these cells makes them potentially sensitive to ER stress .
Recent work suggests that the secretory phenotype of classical PDA is ameliorated by the classical-specific
TF MYRF. MYRF is an ER membrane-associated factor that undergoes autocatalytic cleavage to release an
N-terminal fragment that translocates to the nucleus to regulate transcription [62,63] . Depletion of MYRF
resulted in activation of the transcriptional hallmarks of the unfolded protein response, together with a
massive disruption in ER structure . The mechanisms by which MYRF maintains proper ER function
[64]
appear to be multiple. On the one hand, MYRF knockout led to increased production of mRNAs encoding
secreted factors that are normally highly expressed in classical PDA, such as CEACAM5 and CEACAM6.
While this may seem counterintuitive given that MYRF and the secreted factors are co-expressed in classical
tumors, these authors demonstrated that MYRF binds directly to secreted factor loci and attenuates their
expression, presumably avoiding ER stress that might accompany their overproduction. When MYRF was
introduced into basal-like PDA cells, ER function was improved, which partially occurred using a version of
MYRF lacking its DNA binding domain, suggesting that the membrane-bound form that remains in the ER
may also have effects on ER function that do not involve transcription. Another classical-linked TF that may
contribute to ER homeostasis is SPDEF. SPDEF is an ETS-family TF expressed in secretory Goblet and
Paneth cells in the intestine [65,66] . SPDEF is preferentially expressed in classical PDA cells and promotes the
expression of ER-resident AGR2 and ERN2, two proteins that contribute to ER homeostasis [67-69] . SPDEF
knockout in classical but not basal-like cell lines restrained tumorigenesis, supporting the idea that classical
PDA lines are particularly dependent on pathways that maintain ER function.

Classical TFs as tumor suppressors and dependencies
We have seen that TFs that enforce the classical state are often not only dispensable for tumor development
but even serve as an impediment to carcinogenesis. The fact that TFs linked to maintenance of the classical
state restrain tumorigenesis appears consistent with the better overall survival of patients with classical
compared to basal-like tumors and indicates that PDA subtypes can readily interconvert in response to
experimental TF manipulation. However, the enrichment of GATA6 amplifications in classical tumors
implies that at certain points in the development of these tumors, the high-level expression of GATA6
provides a selective advantage. In that light, experiments showing that depletion of GATA6 can result in a
smooth transition to a basal-like phenotype are somewhat surprising. In fact, classical PDA cells do not
always readily abandon the classical state for basal-like state. KLF5 is repressed during a transition from
[53]
classical to basal-like PDA , which is consistent with the fact that KLF5 is repressed by SMAD TFs
downstream of TGF-β during the EMT . EMT in classical tumor cells can result in cell death and tumor
[70]
[70]
suppression, and experimental depletion of KLF5 can result in the same . In the case of KLF5,
indispensability in classical cells stems from its collaboration with another TF, SOX4. SOX4 normally plays
a tumorigenic role in classical PDA, and binds the genome with a high degree of overlap with KLF5. When
KLF5 is depleted through TGF-β repression during EMT, SOX4 becomes a driver of apoptosis .
[70]
Importantly, activation of certain signaling pathways such as AKT/PI3K can smoothen the transition by
[70]
preventing cell death . In this way, under some conditions, the EMT serves as a bottleneck for classical
PDA cells, but the bottleneck can be “widened” by specific signaling pathways. Successful transit through
this bottleneck may yield tumor cells of the basal-like subtype.

TF determinants of basal-like PDA
Fewer TFs have been implicated in maintenance of the basal-like state. Given the histological and
transcriptomic resemblance of basal-like PDA to squamous cell carcinoma, it is unsurprising that a key
squamous lineage regulator, TP63, plays an important role in determining the basal-like state. TP63 is a