We have previously reported on the isolation and initial characterization of BT/HerR
1.0 cells that are highly resistant to Herceptin 
. Those cells were derived by culturing the Her2-dependent, Herceptin-sensitive BT474 human breast cancer cells in the presence of 1.0 µM Herceptin for 5–6 months. Gene profiling was subsequently performed to determine the full complement of gene expression changes in BT/HerR
clones. That analysis revealed changes in a number of genes that intersect with the PKA signaling pathway as either direct or indirect regulators of PKA activity. These include down-regulation of the gene for PKA-RIIα, a PKA regulatory subunit; down-regulation of PKIγ, an endogenous inhibitor of PKA 
; upregulation of PPP1R1B, which codes for Darpp-32 and its transcriptional variant t-Darpp 
; and down-regulation of PPP1R3C, whose gene product has multiple functions including promotion of protein phosphatase-1 (PP-1) activity and modulation of Darpp-32 function 
. The net effect of these changes is 2- to 4-fold activation of PKA signaling activity in BT/HerR
cells (manuscript submitted).
In the current report, we demonstrate that it is the amino-terminal truncated protein, t-Darpp, that was predominantly overexpressed in BT/HerR
cells selected for Herceptin resistance () and that exogenous overexpression of t-Darpp was sufficient for conferring Herceptin resistance on Her2-positive SK-Br-3 cells (). These results are consistent with the recent report from Belkhiri et al
. using independently derived Herceptin-resistant cell lines and transfected cells 
. Hamel et al
. also demonstrate that t-Darpp overexpression can confer resistance 
. We go on to demonstrate that high expression of the full-length protein, Darpp-32, was able to reverse t-Darpp's effects on Herceptin-resistant cell growth and phospho-Akt levels (). In fact, overexpression of Darpp-32 alone appeared to be deleterious to cell survival, since we were never able to obtain long-term cultures of SK-Br-3 cells that overexpress Darpp-32. These data suggest that Darpp-32 and t-Darpp have antagonistic effects on cell growth and/or survival.
In addition to conferring Herceptin resistance, t-Darpp is also reported to confer resistance to drug-induced apoptosis, apparently via a mechanism that involves CREB activation 
. We also show a relationship between t-Darpp overexpression and CREB activation and this was reversed by co-expression of Darpp-32 (). Although not definitive, the most likely means by which CREB is influenced by t-Darpp/Darpp-32 is via the PKA signaling pathway. This would be consistent with the overall implication of PKA regulatory proteins in Herceptin resistance discussed earlier and the known function of Darpp-32 as a PKA inhibitor 
, but the mechanism by which t-Darpp might influence this pathway is not known. Because the full-length Darpp-32 is both a substrate for phosphorylation by PKA (at Thr-34) and a feedback negative regulator of PKA, it is interesting to speculate that t-Darpp might function by antagonizing Darpp-32's feedback inhibition of PKA, thereby potentiating PKA signaling. The downstream effects of PKA signaling include phosphorylation and activation of full-length Darpp-32 as a PP-1 inhibitor 
. Notably, the Thr-34 phosphorylation site and PP-1 binding domain are missing from t-Darpp, suggesting that its overexpression should not have a direct effect on PP-1 activity.
There could be multiple downstream effects of PP-1 inhibition and/or PKA activation, including the sustained phosphorylation and constitutive activation of Akt that is observed in Herceptin resistant cells. As noted earlier, Belkhiri et al
. found that t-Darpp is anti-apoptotic and promotes resistance to cytotoxic agents, additional effects that could be mediated through PP-1 or more directly through PKA 
. One final observation from our reported microarray data is the down-regulation of PPP1R3C in BT/HerR
1.0 clones. The corresponding gene product, PTG, is a scaffolding protein that can stimulate PP-1 activity and also has been reported to interfere with Darpp-32's inhibition of PP-1 
. Thus, down-regulation of PTG would be consistent with a mechanism that results in overall inhibition of PP-1 and sustained Akt phosphorylation.
Based on the findings in the current report, our previous observations on the role of PKA and its regulatory proteins in Herceptin resistance, and the work of Belkhiri et al
., we propose a working model in which down-regulation of PKA-RIIα, PKIγ and PTG and up-regulation of t-Darpp work in concert to enhance PKA enzyme activity and inhibit PP-1, thereby allowing sustained activation of the PI3K/Akt pathway in the presence of Herceptin, and promoting cell growth and survival. This model is illustrated in . Additional direct or indirect effects of t-Darpp overexpression on Her2 or Akt or other downstream effects of PKA dysregulation, as suggested by Belkhiri et al
, might also contribute to the resistance phenotype. It is worth noting that this same group has not reported the anti-resistance effects of Darpp-32 overexpression that we demonstrate here. In fact, those authors claim that Darpp-32 is able to confer resistance to chemotherapy-induced apoptosis in a colon cancer cell line (RKO) and a gastric cancer cell line (AGS) 
. Interestingly, Darpp-32 (but not t-Darpp) overexpression results in increased basal apoptosis rates in AGS cells and the protection against drug-mediated apoptosis is more pronounced with t-Darpp overexpression than with Darpp-32 overexpression in those cells. This is especially true for ceramide, a drug that activates protein phosphatases such as PP-1 and PP2A, thus indirectly inhibiting Akt signaling. Hamel et al.
also suggest that Darpp-32 is able to confer resistance to Herceptin 
, but there is no direct demonstration of Darpp-32 expression in cells analyzed for the Herceptin phenotype in that study. The possible conflicts or overlaps between our current observations and those of Belkhiri et al
. and Hamel et al
. still need to be resolved, but they could be due to different cell lines used, different levels of exogenous gene expression, or other factors affecting the intracellular function of Darpp-32.
A working model of Herceptin resistance in BT/HerR cells.
There could be a broader role for t-Darpp and/or Darpp-32 in malignancy, beyond an involvement in drug resistance. A protein(s) cross-reacting with Darpp-32 antibody and mRNA corresponding to Darpp-32 and t-Darpp are detected in a variety of adenocarcinomas, including breast cancers [30,33,34]. Using antibodies that detect either Darpp-32 alone or both Darpp-32 and t-Darpp, we have found frequent high-level expression of one or both proteins in both Her2-positive and Her2-negative breast cancers (unpublished observations). Work in the current report would suggest that it is t-Darpp, or perhaps the ratio between t-Darpp and Darpp-32, that is critical for both malignant growth and clinical outcome of these cancers, but further analysis will be required to determine if this is the case. If it is, then t-Darpp (or, more generally, the PKA pathway) could become a new target for therapy and/or a biomarker for response to drug therapy. Moreover, it will be interesting to study the mechanism by which cells regulate expression from the upstream (Darpp-32) and downstream (t-Darpp) transcriptional start sites in the PPP1R1B gene, since shifting the balance towards the t-Darpp start site could have profound effects on both malignancy and drug resistance.